Methods for recording the reaction history of a solid support

ABSTRACT

Encoded combinatorial chemistry is provided, where sequential synthetic schemes are recorded using organic molecules, which define choice of reactant, and stage, as the same or different bit of information. Various products can be produced in the multi-stage synthesis, such as oligomers and synthetic non-repetitive organic molecules. Conveniently, nested families of compounds can be employed as identifiers, where number and/or position of a substituent define the choice. Alternatively, detectable functionalities may be employed, such as radioisotopes, fluorescers, halogens, and the like, where presence and ratios of two different groups can be used to define stage or choice. Particularly, pluralities of identifiers may be used to provide a binary or higher code, so as to define a plurality of choices with only a few detachable tags. The particles may be screened for a characteristic of interest, particularly binding affinity, where the products may be detached from the particle or retained on the particle. The reaction history of the particles which are positive for the characteristic can be determined by the release of the tags and analysis to define the reaction history of the particle.

This is a divisional of U.S. Ser. No. 08/227,007 filed Apr. 13, 1994,now U.S. Pat. No. 5,565,324 which is a continuation-in-part of U.S. Ser.No. 08/159,861, filed Nov. 30, 1993 now abandoned, which is acontinuation-in-part of U.S. Ser. No. 08/130,271, filed Oct. 1, 1993,now abandoned, which is a continuation-in-part of U.S. Ser. No.08/013,948, filed Feb. 4, 1993, now abandoned, which is acontinuation-in-part of U.S. Ser. No. 07/955,371, now abandoned, filedOct. 1, 1992, the contents of which are hereby incorporated by referenceinto the subject application.

TECHNICAL FIELD

The field of this invention concerns combinatorial chemistry whichinvolves syntheses having a plurality of stages, with each stageinvolving a plurality of choices, where large numbers of products havingvarying compositions are obtained.

BACKGROUND OF THE INVENTION

There is substantial interest in devising facile methods for thesynthesis of large numbers of diverse compounds which can then bescreened for various possible physiological or other activities.Typically such a synthesis involves successive stages, each of whichinvolves a chemical modification of the then existing molecule. Forexample, the chemical modification may involve the addition of a unit,e.g. a monomer or synthon, to a growing sequence or modification of afunctional group. By employing syntheses where the chemical modificationinvolves the addition of units, such as amino acids, nucleotides,sugars, lipids, or heterocyclic compounds where the units may benaturally-occurring, synthetic, or combinations thereof. One may createa large number of compounds. Thus, even if one restricted the synthesisto naturally-occurring units or building blocks, the number of choiceswould be very large, 4 in the case of nucleotides, 20 in the case of thecommon amino acids, and essentially an unlimited number in the case ofsugars.

One disadvantage heretofore inherent in the production of large numberof diverse compounds, where at each stage of the synthesis there are asignificant number of choices, is the fact that each individual compoundwill be present in a minute amount. While a characteristic of aparticular compound, e.g. a physiological activity, may be determinable,it is usually impossible to identify the chemical structure of thisparticular compound present.

Moreover, physiologically-active compounds have historically beendiscovered by assaying crude broths using Edisonian or stochastictechniques, where only a relatively few compounds are assayed at a time,or where a limited number of structural similar homologs ofnaturally-occurring physiologically-active compounds are assayed. Two ofthe major problems has been associated with the use of such crudebroths, namely, the necessity to purify the reaction mixture intoindividual component compounds and the time-consuming effort required toestablish the structure of the compound once purified.

To address these disadvantages and problems, techniques have beendeveloped in which one adds individual units as part of a chemicalsynthesis sequentially, either in a controlled or a random manner, toproduce all or a substantial proportion of the possible compounds whichcan result from the different choices possible at each sequential stagein the synthesis. However, for these techniques to be successful it isnecessary for the compounds made by them to be amenable to methods whichwill allow one to determine the composition of a particular compound somade which shows a characteristic of interest.

One such approach involves using a chip which allows for separateanalysis at physically separate sites on the surface of the chip (Fodoret al., Science 251: 767 [1991]). By knowing what reactant is addedsequentially at each such site, one can record the sequence of eventsand thus the series of reactions. If one then subjects the chip to ascreening method for a particular desired characteristic and detects thecharacteristic one can really determine the compound synthesized at thesite which demonstrates that characteristic.

Another such technique involves the theoretical synthesis ofoligonucleotides in parallel with the synthesis of oligopeptides as thecompounds of interest (Brenner and Lerner, PNAS USA [1992] 81:5381-5383).

Further techniques are also disclosed in the following publications:Amoto, Science (1992) 257, 330-331 discusses the use of cosynthesizedDNA labels to identify polypeptides. Lam, et al., Nature (1991) 354,82-84 describe a method for making large peptide libraries. Houghton, etal., Nature (1991) 354, 84-86 and Jung and Beck-Sickinger, Angew. Chem.Int. Ed. Engl. (1992) 91, 367-383 describe methodology for making largepeptide libraries. Kerr et al., J. Amer. Chem. Soc., (1993) 115, 2529-31teach a method of synthesizing oligomer libraries encoded by peptidechains.

However, since methods such as the preceding typically require theadditum of like moieties, there is substantial interest in discoveringmethods for producing compounds which are not limited to sequentialaddition of like moieties. Such methods would find application, forexample, in the modification of steroids, antibiotics, sugars,coenzymes, enzyme inhibitors, ligands and the like, which frequentlyinvolve a multi-stage synthesis in which one would wish to vary thereagents and/or conditions to provide a variety of compounds. In suchmethods the reagents may be organic or inorganic reagents, wherefunctionalities may be introduced or modified, side groups attached orremoved, rings opened or closed, stereochemistry changed, and the like.(See, for example, Bunin and Ellman, JACS 114, 10997 [1992].) For such amethod to be viable, however, there needs to be a convenient way toidentify the structures of the large number of compounds which resultfrom a wide variety of different modifications. Thus, there is a need tofind a way whereby the reaction history may be recorded, and desirably,the structures of the results compound identified.

Finally as the size of a library compounds so synthesized increases,known techniques of structure elucidation and product segregationintroduce substantial inefficiencies and uncertainties which hinder theaccurate determination of the structure of any compound identified asbeing of interest. Thus, there is a substantial need for new methodswhich will permit the synthesis of complex combinatorial chemicallibraries which readily permit accurate structural determination ofindividual compounds within the library which are identified as being ofinterest.

Finally, international applications WO91/17823 and WO92/09300 concerncombinatorial libraries.

Many of the disadvantages of the previously-described methods as well asmany of the needs not met by them are addressed by the present inventionwhich, as described more fully hereinafter, provides marlad advantagesover these previously-described methods.

SUMMARY OF THE INVENTION

Methods and compositions are provided for encoded combinatorialchemistry, whereby at each stage of the synthesis, a support such as aparticle upon which a compound is being synthesized is uniquely taggedto define a particular event, usually chemical, associated with thesynthesis of the compound on the support. The tagging is accomplishedusing identifier molecules which record the sequential events to whichthe supporting particle is exposed during synthesis, thus providing areaction history for the compound produced on the support.

Each identifier molecule is characterized by being stable under thesynthetic conditions employed, by remaining associated with the supportsduring the stage of the synthesis, by uniquely defining a particularevent during the synthesis which reflects a particular reaction choiceat a given stage of the synthesis, by being distinguishable from othercomponents that may be present during assaying, and by allowing fordetachment of a tag component which is discernible by a convenient,analytical technique.

The identifiers of this invention are used in combination with oneanother to form a binary or higher order encoding system permitting arelatively small number of identifiers to be used to encode a relativelylarge number of reaction products. For example, when used in a binarycode N identifiers can uniquely encode up to 2^(N) different compounds.

Moreover, the identifiers of this invention need not be bound seriallythrough a previous identifier but rather are individually bound to thesubstrate, either directly or through the product being synthesized. Theidentifiers are not sequencable. Furthermore, the identifiers contain acleavable member or moiety which permits detachment of a tag componentwhich can be readily analyzed.

Conveniently, the combinatorial synthesis employs definable solidsupports upon which reactions are performed and to which the identifiersare bound. The individual solid supports or substrates carrying thefinal product compounds may be screened for a characteristic of interestand the reaction history determined by analyzing the associatedidentifier tags.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the analysis of tag 4 by mass spectroscopy. Twosignals corresponding to tag 4 were observed.

FIG. 2 illustrates the analysis of tag 11 by mass spectroscopy. Twosignals corresponding to tag 11 were observed.

FIG. 3 illustrates the analysis of tag 13 by mass spectroscopy. Twosignals corresponding to tag 13 were observed.

FIG. 4 illustrates the analysis of tags 4, 11 and 13 by positivechemical ionization mass spectroscopy (PCIMS) when approximately equalamounts of each tag were mixed together. Two signals corresponding toeach separate tag could easily be distinguished.

DETAILED DESCRIPTION OF THE INVENTION

As used in this application the term "tag" or "T" means a chemicalmoiety which possesses two properties. First, it is capable of beingdistinguished from all other chemical moieties. Second, it is capable ofbeing detected when present at 10⁻¹⁸ to 10⁻⁹ mole. These two propertiesmay be embodied in a single chemical structure. Alternatively, theseproperties may be embodied in separate chemical structures which arelinked together. In this latter case, one of the chemical structures,which may be designated C (or in the case of more than one suchstructure C, C', etc.) provides the property of rendering the tagdistinguishable from other tags while the other chemical structure, E,provides the property of rendering the tag detectable and optionally mayprovide the property of rendering the tag separable from other tags.

As used in this application, the term "linker" or "L" means a chemicalmoiety which possesses three properties. First, it is attachable to asolid support. Second, it is attachable to a tag. Third, when it isattached to both a solid support and a tag, it is cleavable such thatthe tag may be released from the solid support. These three propertiesmay be embodied in a single chemical structure. Alternatively, theseproperties are embodied in three chemical structures which are linkedtogether. In this latter case one of the chemical structures, which maybe designated F¹, provides the property of rendering the linkerattachable to the solid support; the second chemical structure, whichmay be designated V, provides the property of rendering the linkercleavable; and the third chemical structure which may be designed A',provides the property of rendering the linker attachable to the tag.Desirably, the chemical structures V and A' are one and the same, inwhich case V-A' may be designated F².

As used in this application, the term "identifier" means a chemicalentity which includes both a tag and a linker. Thus, in the broadestsense an identifier may be represented by the formula L-T while specificembodiments of the identifier may be represented by the formulaeF'-V-A'-T; F¹ -V-A'-C-E (or F¹ -V-A'-E-C); L-C-E (or L-E-C); andL-C-E-C'.

As used in this application, the term "bound identifier" means anidentifier attached to a solid support.

As used herein, the term "choice" means the alternative variables for agiven stage in a combinatorial synthesis, such as reactant, reagent,reaction conditions, and combinations thereof. The term "stage"corresponds to a step in the sequential synthesis of a compound orligand; the compound or ligand being the final product of acombinatorial synthesis.

The term "alkyl" includes linear, branched, and cyclic structures andcombinations thereof. Thus, the term includes methyl, ethyl, propyl,isopropyl, butyl, sec- and tert-butyl, cyclopropyl, cyclobutyl,cyclopentyl, 2-methylcyclopropyl, and the like. Lower alkyl is C₁ -C₆alkyl. Lower alkenyl is C₂ -C₆ alkenyl of a linear, branched, or cyclicconfiguration and combinations thereof.

Unless otherwise indicated, it is intended that the definitions of anysubstituent (e.g., R¹ R², Z, etc.) in a particular molecule beindependent of its definitions elsewhere in the molecule. Thus, NR⁴ R⁴represents NHH, NHCH₃, NHCH₂ CH₃, N(CH₃)₂, etc.

Some of the compounds described herein contain one or more centers ofasymmetry and may thus give rise to enantiomers, diastereoisomers, andother steroisomeric forms. The present invention is meant to include allsuch possible stereoisomers as well as their racemic and optically pureforms. Optically active (R) and (S) isomers may be prepared using chiralsynthons, chiral reagents, or resolved using conventional techniques.When the compounds described herein contain olefinic double bonds, it isintended to include both E and Z geometric isomers.

The materials upon which the combinatorial syntheses of this inventionare performed are referred to herein interchangeably as beads, solidsurfaces, (solid) substrates, particles, supports, etc. These terms areintended to include:

a) solid supports such as beads, pellets, disks, capillaries, hollowfibers, needles, solid fibers, cellulose beads, pore-glass beads, silicagels, polystyrene beads optionally cross-linked with divinylbenzene,grafted co-poly beads, poly-acrylamide beads, latex beads,dimethylacrylamide beads optionally cross-linked with N,N'-bis-acryloylethylene diamine, glass particles coated with a hydrophobic polymer,etc., i.e., a material having a rigid or semi-rigid surface; and

b) soluble supports such as low molecular weight non-cross-linkedpolystyrene.

These materials must contain functionalities or must be able to befunctionalized such that identifiers or product intermediates may beattached to them.

In addition, the following abbreviations have the indicated meanings:##EQU1##

The subject invention concerns the production of libraries of products,i.e. compounds, where the individual products or compounds present inthe libraries may be physically separated from one another and may bescreened for a characteristic of interest either bound to, or detachedfrom, a solid support. By having serial syntheses, where at each stageof a synthesis each of the individual intermediates is treated in avariety of ways, a very large number of products is produced, each ofwhich is present in a small amount, frequently less than 100 pmol, morefrequently less than 10 nmol. Because of the small quantity of finalproduct or compound so produced, identifying these products by isolatingand structurally elucidating the products would generally not befeasible. Moreover, in sequential synthesis involving other than theaddition of similar units, the analysis would be arduous if notimpossible using the amount of product typically available. However, byassociating each choice or combination of choices (e.g., "add reagent A"or "add reagent A, then reagent B, and heat to 100° C. for 2 hrs.") ofthe serial synthesis with a combination of identifiers which define thechoice of variables such as reactant, reagent, reaction conditions, or acombination of these, one can use the identifiers to define the reactionhistory of each definable and separable substrate. The analysis of tagsdetached from the identifiers allows for ready identification of thereaction history, at picomolar or lower concentrations, e.g. femtomolaror less. One can determine a characteristic of a product of a synthesis,usually a chemical or biological characteristic by various screeningtechniques, and then identify the reaction history and thereby thestructure of that product, which has the desired characteristic, byvirtue of the tags associated with the product.

The use of the instant multiple tag system avoids the necessity ofcarrying out a complicated cosynthesis which reduces yields and requiresmultiple protecting groups, and avoids the necessity of usingsequencable tags which are necessarily chemically labile. Both thenecessity of multiple protecting groups and the intrinsic instability ofall known sequencable tagging molecules (i.e., nucleic acid or peptideoligomers) severely limit the chemistry which may be used in thesynthesis of the library element or ligand.

Moreover, the use of a binary, or higher order, multiple tag systemreduces enormously the number of tags necessary to encode thereagent/reactant choice in any stage in a synthesis. For example, if aparticular synthetic stage could be carried with 125 different choicesfor reagent, the binary system would require only 7 tags. This can makethe difference between a practical encoding system and an impracticalone, because it may not be feasible to obtain and use the large numberof distinguishable tags required by other systems. With the binarysystem of the invention, 30 distinguishable tags are available and aresufficient to encode >10⁹ different syntheses.

Importantly, the present method employs tags which are detachable from aligand or compound synthesized also for the purpose of decoding. Suchdetachability also allows the tags to be distinguished on more than onebasis; in particular, they can be separated (e.g., on the basis ofchromatographic retention time) and then analyzed (e.g., a second basisis a spectral property such as mass spectroscopy m/e, orelectrophoricity). Having multiple bases for distinction allows theencoding of large amounts of information with a small number of tags.

Detachment further allows tags to be detected at very low levels,because they can be removed from the support matrix on which thesynthesis is effected and from the ligand synthesized, the presence ofeither of which could provide spurious background signals, e.g. byquenching fluorescence or the like.

Detachable tags are also amenable to rapid analysis by automatedsampling systems, and allow for selective derivatization for detectionvia functional groups, eliminating any incompatibility between thedetection moiety and the reaction conditions used in the synthesis.

Inherent in any tagging scheme is the requirement that the chemicalcharacteristics of the tags and the chemical stages for theirincorporation be compatible with the characteristics of the ligand andthe stages in their synthesis, and vice versa. The advantage of tagsthat are generally unreactive, as exemplified hereinafter by thesubstituted-aryloxypolymethylene moieties, is a greater range ofchemical transformations and chemical functionality that can be employedin synthesis of the ligands.

A further advantage of the chemically stable tags of this invention istheir compatibility with a greater variety of rapid, convenient-methodsof separation and analysis, such as gas chromatography and massspectrometry. Moreover, the organic tags of this inventions generally donot specifically interact with biological receptors. Thus, these tagswill generally not give spurious results in biological assays and willgenerally not be modified by enzymes or other biological molecules.

Finally, the chemical stability of the present tags allows them to bedetached by a wide variety of methods which improves sensitivity intheir analysis as described above.

Thus, this invention provides methods and compositions for encodedcombinatorial synthesis whereby at each stage of the synthesis one ormore identifiers are provided which encode an event associated with aparticle stage in the synthesis of a compound on a support or particle.This event comprises the choice of reactant and/or reaction conditionsat that stage of the reactions where each such stage may involve one ormore reactants which are the same or different under the same ordifferent conditions, e.g. partial reactions, multiple additions, rateof addition, differing combinations of reagents, etc. In addition,groups of particles may be sequestered from other groups of particlesand subjected to a different series of events at any time during thecourse of the sequential synthesis.

By providing N identifiers, each having M distinguishable states, M^(N)different syntheses can be uniquely defined. In the case of M=2 wherethe two states could be the presence or absence of identifier, thesynthesis would thus be defined by a base 2 or binary code. In the caseof M=3 where the three states could be the presence of an identifier attwo distinguishable concentrations or its absence, the synthesis wouldbe defined by a base 3 code. Herein, such base M codes where M>2 aretermed higher order codes. The advantage of higher order codes over abinary code is that fewer identifiers are required to encode the samequantity of information about the synthesis. The products which areproduced will be defined as resulting from a serial synthesis. At eachstage in the synthesis, there is available a plurality of reactantsand/or reagents and/or conditions, which result in a feature of theproduct in relation to an identifiable and usually separable entity,e.g. tag. In referring to reactants and reagents, it is intended thatthe reactant, for the most part, becomes incorporated into the product,e.g. an amino acid, nucleotide, nucleophile, electrophile, diene,alkylating or acylating agent, diamine, or any other synthon, etc. whilea reagent may or may not become incorporated into the product, e.g.base, acid, heat, oxidizing or reducing agent, while both will beincluded under the term "agent". The synthesis may involve individualreactants which become incorporated into the product. Alternatively, astage may involve one or more reactions which result in a modificationof a reaction intermediate. In many cases, combinations of thesepossibilities will be involved.

Using a base 2 or binary code (M=2) and three identifiers (N=3), as manyas 8 (2³) agents for a given stage in a synthesis may be encoded. If thethree identifiers are represented as T1, T2, and T3 and the presence orabsence of each identifier is represented as a `0` or `1` respectively,then eight different agents could be represented in a binary code asfollows:

    ______________________________________                                                  Agent 1 Agent 2    Agent 3                                                                             Agent 4                                      T1,T2,T3 0,0,0 1,0,0 0,1,0 1,1,0                                               Agent 5 Agent 6 Agent 7 Agent 8                                              T1,T2,T3 0,0,1 1,0,1 0,1,1 1,1,1                                            ______________________________________                                    

Similarly, even more information about the synthesis may be encoded bymore identifiers. For example, 9 identifiers (N=3) and a base 2 code(M=2) would allow up to 2⁹ or 512 different agent choices to be encoded.Using a base 3 code (M=3) and three identifiers (N=3) would allow asmany as 27 (3³) agent choices to be encoded. If the three identifiersare represented as T1, T2 and T3, and the absence of an identifier isrepresented as a `0`, its presence at a quantity of -0.5 pmol/bead as a`1`, and its presence at a quantity of -1.0 pmol/bead as a `2`, then the27 different agents could be represented by three identifiers in base 3code as:

    ______________________________________                                                  Agent 1 Agent 2    Agent 3                                                                             Agent 4                                      T1,T2,T3 0,0,0 1,0,0 2,0,0 0,1,0                                               Agent 5 Agent 6 . . . Agent 27                                               T1,T2,T3 0,0,1 2,1,0 . . . 2,2,2                                            ______________________________________                                    

To make such higher order encoding schemes practical, one additionalidentifier at a given quantity (e.g., -1.0 pmol/bead) would be added toall members of the library to provide a standard against which thequantities of all identifiers would be measured. The quantities of theidentifiers could be measured by gas chromatography or HPLC with avariety of detection methods. In the case of HPLC, quantities could beconveniently measured by scintillation counting if the identifiers wereradioactively labeled by different quantities of a radionuclide such astritium (³ H). It would be particularly convenient to carry out thequantitation by measuring the ³ H-to-¹⁴ C ratio, thus using ¹⁴ C as astandard. In this way, as many as ten quantities of ³ H could bedistinguished to create a base 10 or decimal code (M=10) which couldencode enormous amounts of information with very few identifiers.

Products and Synthetic Strategies

For the most part, the products of the method of this invention will beorganic compounds where the serial synthesis will involve the additionor removal of chemical units, reactions involving the modification orintroduction of one or more functionalities, ring openings, ringclosings, etc. Chemical units can take many forms, bothnaturally-occurring and synthetic, such as nucleophiles, electrophiles,dienes, alkylating or acylating agents, diamines, nucleotides, aminoacids, sugars, lipids, or derivatives thereof, organic monomers,synthons, and combinations thereof. Alternatively, reactions may beinvolved which result in alkylation, acylation, nitration, halogenation,oxidation, reduction, hydrolysis, substitution, elimination, addition,and the like. This process can produce non-oligomers, oligomers, orcombinations thereof in extremely small amounts, where the reactionhistory, and composition in appropriate cases, can be defined by thepresent tags. Non-oligomers include a wide variety of organic molecules,e.g. heterocyclics, aromatics, alicyclics, aliphatics and combinationsthereof, comprising steroids, antibiotics, enzyme inhibitors, ligands,hormones, drugs, alkaloids, opioids, terpenes, porphyrins, toxins,catalysts, as well as combinations thereof. Oligomers includeoligopeptides, oligonucleotides, oligosaccharides, polylipids,polyesters, polyamides, polyurethanes, polyureas, polyethers, poly(phosphorus derivatives) e.g. phosphates, phosphonates, phosphoramides,phosphonamides, phosphites, phosphinamides, etc., poly (sulfurderivatives) e.g. sulfones, sulfonates, sulfites, sulfonamides,sulfenamides, etc., where for the phosphorous and sulfur derivatives theindicated heteroatom for the most part will be bonded to C, H, N, O orS, and combinations thereof.

Reactions may involve modifications at a variety of random sites of acentral core molecular structure or modifications at a specific site.For example, one may brominate a polycyclic compound, where brominationmay occur at a plurality of sites or use a brominating agent which willbe specific for a particular site, e.g., N-bromosuccinimide. For themost part, reactions will involve single sites or equivalent sites, forexample, one of two hydroxyl groups of a glycol.

For the most part, the subject synthesis will have at least two stageswhere other than bifunctional compounds are attached using the samelinking functionality, e.g. amino acids and amide bonds, nucleotides andphosphate ester bonds, or mimetic compounds thereof, e.g.,aminoiso-cyanates and urea bonds.

The methods of the invention permit variation in reaction at each stage,depending on the choice of agents and conditions involved. Thus, foramino acids, one may have up to 20 amino acids involved using the commonnaturally-encoded amino acids and a much wider choice, if one wishes touse other amino acids, such as D-amino acids, amino acids having theamino group at other than the α-position, amino acids having differentsubstituents on the side chain or substituents on the amino group, andthe like. For the different nucleic acids, there will usually be up to 4natural nucleic acids used for either DNA or RNA and a much largernumber is available if one does not choose to use those particularnucleic acids. For the sugars and lipids, there are a very large numberof different compounds, which compounds may be further increased byvarious substitutions, where all of these compounds may be used in thesynthesis. For individual organic compounds the choice may beastronomically large. In addition, one may have mimetic analogs, whereureas, urethanes, carbonylmethylene groups, and the like may substitutefor the peptide linkage; various organic and inorganic groups maysubstitute for the phosphate linkage; and nitrogen or sulfur maysubstitute for oxygen in an ether linkage or vice versa.

The synthetic strategies will vary with the nature of the group ofproducts one wishes to produce. Thus, the strategy must take intoconsideration the ability to stage-wise change the nature of theproduct, while allowing for retention of the results of the previousstages and anticipating needs for the future stages. Where the variousunits are of the same family, such as nucleotides, amino acids andsugars, the synthetic strategies are relatively well-established andfrequently conventional chemistry will be available. Thus, fornucleotides, phosphoramidite or phosphite chemistries may be employed;for oligopeptides, Fmoc or Boc chemistries may be employed whereconventional protective groups are used; for sugars, the strategies maybe less conventional, but a large number of protective groups, reactivefunctionalities, and conditions have been established for the synthesisof polysaccharides. For other types of chemistries, one will look to thenature of the individual unit and either synthetic opportunities will beknown or will be devised, as appropriate.

In some instances, one may wish to have the same or different blocksintroduced at the same or different stages. For example, one may wish tohave a common peptide functional unit, e.g. the fibronectin binding unit(RGDS), a polysaccharide, e.g. Le^(x), an organic group, e.g. a lactam,lactone, benzene ring, olefin, glycol, thioether, etc. introduced duringthe synthesis. In this manner one may achieve a molecular context intowhich the variation is introduced. These situations may involve only afew stages having the plurality of choices, where a large number ofproducts are produced in relation to a particular functional entity.This could have particular application where one is interested in alarge number of derivatives related to a core molecule or unit known tohave a characteristic of interest.

In developing synthetic strategies, one can provide for batch synthesisof a few compounds which would be prepared during the course of thecombinatorial synthesis. By taking extreme examples, for example,syntheses which might involve steric hindrance, charge and/or dipoleinteractions, alternative reaction pathways, or the like, one canoptimize conditions to provide for enhanced yields of compounds whichmight not otherwise be formed or be formed only in low yield. In thismanner, one may allow for a variety of reaction conditions during thecombinatorial synthesis, involving differences in solvent, temperatures,times, concentrations, and the like. Furthermore, one may use the batchsyntheses, which will provide much higher concentrations of particularproducts than the combinatorial synthesis, to develop assays tocharacterize the activity of the compounds.

Supports: Attachment and Detachment

The synthetic protocol requires that one provide for a plurality ofdifferent reactions involving different reactants resulting in aplurality of different intermediates at each stage of the synthesis.While other techniques are available, this can be achieved mostconveniently by employing small definable solid substrates, commerciallyavailable as beads, which can be readily mixed, separated, and serve asa solid substrate for the sequential synthesis. The solid substrates maybe solid, porous, deformable or hard, and have any convenient structureand shape. In some instances, magnetic or fluorescent beads may beuseful. The beads will generally be at least 10-2000 μm, usually atleast 20-500 μm, more usually at least 50-250 μm in diameter.

Any convenient composition can be used for the particles or beads, whichbead composition will maintain its mechanical integrity during thevarious process stages, can be functionalized, has functional groups orallows for reaction with an active species, allows for the serialsynthesis as well as attachment of the identifiers, can be readily mixedand separated, and will allow for convenient detachment of the tags andproducts. Beads which may be employed include cellulose beads,pore-glass beads, silica gel, polystyrene beads, particularlypolystyrene beads cross-linked with divinylbenzene, grafted co-polymerbeads such as polyethyleneglycol/polystyrene, polyacrylamide beads,latex beads, dimethylacrylamide beads, particularly cross-linked withN,N'-bis-acryloyl ethylene diamine and comprisingN-t-butoxycarbonyl-β-alanyl-N'-acryloyl hexamethylene diamine,composites, such as glass particles coated with a hydrophobic polymersuch as cross-linked polystyrene or a fluorinated ethylene polymer towhich is grafted linear polystyrene; and the like. General reviews ofuseful solid supports (particles) that include a covalently-linkedreactive functionality may be found in Atherton, et al., Prospectives inPeptide Chemistry, Karger, 101-117 (1981); Amamath, et al., Chem. Rev.77:183-217 (1977); and Fridkin, The Peptides, Vol. 2, Chapter 3,Academic Press, Inc., (1979), pp. 333-363.

Depending upon the nature of the synthetic procedure or the assay of thefinal product, one or another bead may be more or less desirable. Whilebeads are especially convenient, other solid supports may also find use,such as capillaries, hollow fibers, needles, solid fibers, etc., wherethe size of the solid support allows for the desired variation inreaction histories.

Depending upon the nature of the synthesis, the beads may befunctionalized in a variety of ways to allow for attachment of theinitial reactant. These may be linked through a non-labile linkage suchas an ester bond, amide bond, amine bond, ether bond, or through asulfur, silicon, or carbon atom, depending upon whether one wishes to beable to remove the product from the bead. Conveniently, the bond to thebead may be permanent, but a linker between the bead and the product maybe provided which is cleavable such as exemplified in Table 1. Two ormore different linkages may be employed to allow for differentialrelease of tags and/or products.

Depending upon the nature of the linking group bound to the particle,reactive functionalities on the bead may not be necessary where themanner of linking allows for insertion into single or double bonds, suchas is available with carbenes and nitrenes or other highly-reactivespecies. In this case, the cleavable linkage will be provided in thelinking group which joins the product or the tag to the bead.

Desirably, when the product is permanently attached, the link to thebead will be extended, so that the bead will not sterically interferewith the binding of the product during screening. Various links may beemployed, particular hydrophilic links, such as polyethyleneoxy,saccharide, polyol, esters, amides, combinations thereof, and the like.

Functionalities present on the bead may include hydroxy, carboxy,iminohalide, amino, thio, active halogen (Cl or Br) or pseudohalogen(e.g., --CF₃, --CN, etc.), carbonyl, silyl, tosyl, mesylates,brosylates, triflates or the like. In selecting the functionality, someconsideration should be given to the fact that the identifiers willusually also become bound to the bead. Consideration will includewhether the same or a different functionality should be associated withthe product and the identifier, as well as whether the twofunctionalities will be compatible with the product or identifierattachment and tag detachment stages, as appropriate. Different linkinggroups may be employed for the product, so that a specific quantity ofthe product may be selectively released. In some instances the particlemay have protected functionalities which may be partially or whollydeprotected prior to each stage, and in the latter case, reprotected.For example, amino may be protected with a carbobenzoxy group as inpolypeptide synthesis, hydroxy with a benzyl ether, etc.

Where detachment of the product is desired, there are numerousfunctionalities and reactants which may be used. Conveniently, ethersmay be used, where substituted benzyl ether or derivatives thereof, e.g.benzhydryl ether, indanyl ether, etc. may be cleaved by acidic or mildreductive conditions. Alternatively, one may employ β-elimination, wherea mild base may serve to release the product. Acetals, including thethio analogs thereof, may be employed, where mild acid, particularly inthe presence of a capturing carbonyl compound, may serve. By combiningformaldehyde, HCl and an alcohol moiety, an α-chloroether is formed.This may then be coupled with an hydroxy functionality on the bead toform the acetal. Various photolabile linkages may be employed, such aso-nitrobenzyl, 7-nitroindanyl, 2-nitrobenzhydryl ethers or esters, etc.Esters and amides may serve as linkers, where half-acid esters or amidesare formed, particularly with cyclic anhydrides, followed by reactionwith hydroxyl or amino functionalities on the bead, using a couplingagent such as a carbodiimide. Peptides may be used as linkers, where thesequence is subject to enzymatic hydrolysis, particularly where theenzyme recognizes a specific sequence. Carbonates and carbamates may beprepared using carbonic acid derivatives, e.g. phosgene, carbonyldiimidazole, etc. and a mild base. The link may be cleaved using acid,base or a strong reductant, e.g., LiAlH₄, particularly for the carbonateesters. For a list of cleavable linkages, see, for example, Greene andWuts, Protective Groups in Organic Synthesis, 2nd ed. Wiley, 1991. Theversatility of the various systems that have been developed allows forbroad variation in the conditions for attachment of products andidentifiers and differential detachment of products and tags, asdesired.

The following table indicates various illustrative linking units (i.e.,F² in Formula I) and the manner in which they may be cleaved:

                                      TABLE 1                                     __________________________________________________________________________    Various illustrative linking units and the                                      manner in which they may be cleaved                                              Linking Group   Cleavage Reagent                                         __________________________________________________________________________      silyl fluoride or acid                                                        A hν                                                                       B Ce(NH.sub.4).sub.2 (NO.sub.3).sub.6                                         --NCO.sub.2 (L)* OH.sup.-, H.sup.+, or LiAlH.sub.4                            C O.sub.3, OsO.sub.4 /IO.sub.4.sup.-, or KMnO.sub.4                           D 1) O.sub.2 or Br.sub.2, MeOH                                                 2) H.sub.3 O.sup.+                                                           --Si--(L) oxidation, H.sup.+, Br.sub.2, Cl.sub.2,                              etc.                                                                         E H.sub.3 O.sup.+                                                             F H.sub.3 O.sup.+                                                             G F.sup.-  or H.sup.+                                                         H base, OH.sup.-                                                              x = keto, ester, amide,                                                       NO.sub.2, sulfide, sulfoxide,                                                 sulfone, and related                                                          electron withdrawing                                                          groups                                                                        I H.sub.3 O.sup.+  or reduction (e.g.                                          Li/NH.sub.3)                                                                 J (φ.sub.3 P).sub.3 RhCl(H)                                               K Li, Mg, or BuLi                                                             M Hg.sup.+2                                                                   N Zn or Mg                                                                    x = halogen or                                                                pseudohalogen                                                                 O oxidation (e.g. Pb(OAc).sub.4                                                or H.sub.5 IO.sub.6)                                                         P base                                                                        x = electron withdrawing                                                      group                                                                       __________________________________________________________________________    *(L) shows the point of attachment of the tag or product.                                       1  STR1##                                                                     2  STR2##                                                                   3  STR3##                                                                     4  STR4##                                                                                5  STR5##                                                                     6  STR6##                                                                     7  STR7##                                          8  STR8##                                                                     9  STR9##                                                                                     0  STR10##                                                                    1  STR11##                                                    2  STR12##                                                                    3  STR13##                                                                    4  STR14##                                                                    5  STR15##                                                                    6  STR16##                                                                    7  STR17##                                                                    8  STR18##                                                                    9  STR19##                                                                                              0  STR20##                                                                    1  STR21##                                                                    2  STR22##                                            L is the tag or product either directly bonded to the indicated atom or                               indirectly bonded through a linking group such                                as C(O)O, which linking group may provide a     

R is H or lower alkyl.

Linker

The choice of linker for the ligand will be part of the syntheticstrategy, since the linking group may result in a residual functionalityon the product. It will usually be difficult, but feasible, to furthermodify the product after detachment from the bead. In designing thesynthetic strategy, one can use a functionality to be retained in theproduct as the point of attachment for the linking group. Alternatively,when permitted by the nature of the product, one could use a cleavage ordetachment method which removes the linking functionality, e.g., anarylthioether or silyl with a metal hydride or acid. Since in many casesthe synthetic strategy will be able to include a functionalized site forlinking, the functionality can be taken advantage of in choosing thelinking group. In some instances it may be desirable to have differentfunctionalities at the site of linking the product to the support, whichmay necessitate using different modes of linking, which modes mustaccommodate either the same detachment method or different detachmentmethods which may be carried out concurrently or consecutively, e.g.,irradiation with light and acid hydrolysis.

Of particular interest for binding the identifiers to the particle arecarbenes and nitrenes which can insert between a carbon and hydrogenatom to form a covalent bond, or into an olefinic bond to form acyclopropane (in the case of carbene) or an aziridine (in the case ofnitrene).

With carbene or nitrene linking groups various substituted benzenes maybe used, where the benzene is substituted with a group capable ofproviding a carbene: CHN₂, COCHN₂, SO₂ CHN₂ ; or nitrene: N₃, NO₂, NO,SO₂ N₃. The carbenes may be generated from diazoalkane derivatives byphotolysis, thermolysis, or by treatment with low valent transitionmetal species, e.g., Rh(OAc)₂. The nitrene may be generated byphotolysis or thermolysis from azides; and from nitro, nitroso andazides by using tervalent phosphorus compounds or low valent transitionmetals.

A group of linker moieties (F¹ -F² -) of particular interest include2-nitro-4-carboxybenzyloxy, 2-nitro-4-diazoacetylbenzyloxy, 4 or 5azidomethylcarbonyl-2-methoxyphenoxy, and 2-methoxy-4, or5-carboxyphenoxy moieties.

Illustrative compounds where T represents the tag, Z represents acarbene or nitrene precursor or a carboxy group, and R is H or loweralkyl are as follows. For photochemical tag detachment (e.g., withultraviolet light at about 350 nm): T 3-Z-2-nitrobenzyl ether, T4-Z-2-nitrobenzyl ether, T 5-Z-2-nitrobenzyl ether, T 6-Z-2-nitrobenzylether, T 2-Z-4-nitrobenzyl ether, T 3-Z-4-nitrobenzyl ether, T3-Z-2-nitrobenzyl carbonate, T 4-Z-2-nitrobenzyl carbonate, T5-Z-2-nitrobenzyl carbonate, T 6-Z-2-nitrobenzyl carbonate, T2-Z-4-nitrobenzyl carbonate, and T 3-Z-4-nitrobenzyl carbonate. Foroxidative detachment (e.g., using ceric ammonium nitrate):1-OT-2-OR-3-Z-benzene, 1-OT-2-OR-4-Z-benzene, 1-OT-2-OR-5-Z-benzene,1-OT-2-OR-6-Z-benzene, 1-OT-4-OR-2-Z-benzene, and 1-OT-4-OR-3-Z-benzene.For reductive or alkylative detachment (e.g. with lithium/ammonia ormethyl iodide): T (2-Z-phenyl)thioether, T (3-Z-phenyl)thioether, and T(4-Z-phenyl)thioether. For desilylative detachment (e.g., usingtetrabutyl ammonium fluoride or acid): T dialkyl-(2-Z-phenyl)silylether, T dialkyl-(3-Z-phenyl)silyl ether, T dialkyl-(4-Z-phenyl)silylether, T-dialkyl-(2-Z-phenyl)silane, T-dialkyl-(3-Z-phenyl)silane, andT-dialkyl-(4-Z-phenyl)silane.

Combinatorial Synthesis

The synthesis will usually involve stages involving at least 2 choices,frequently at least 4 choices, and may involve 10 choices or more.Generally, the number of choices per stage will not exceed about 100,more usually not exceed about 50. The number of stages will usually beat least about 3, more usually at least about 4, frequently at least 5,and not more than about 30, more usually not more than about 25,preferably not more than about 20, more preferably not more than about10, frequently not more than about 8.

The number of choices and stages will usually result in at least anumber of compounds which allows for a sufficient variety to provide areasonable likelihood that at least one compound will have thecharacteristic of interest. The subject methodology allows for producinggreater than 25,000 compounds, usually greater than 50,000 compounds,preferably greater than 200,000 compounds, and a million or more may beproduced. This will usually mean at least 20 compounds but may be 10⁶ ormore.

In some syntheses, a stage may only involve one or two choices, but thissituation will usually be limited in relation to the number of compoundsone wishes to produce and the particular synthetic strategy. In many ofthe strategies, the restricted number of choices, i.e., fewer than 5choices, more usually 2 or fewer choices, will be limited to the greaterof 40% of the total number of stages or about 2 stages in the sequentialsynthesis, more usually limited to 20% of the total number of stages.

Reaction Procedure

In carrying out the synthesis, one may initially begin with a number ofbeads, usually at least 10³, more usually at least 10⁴, and desirably atleast 10⁵, while generally not exceeding at least 10¹⁵, more usually notexceeding at least 10¹⁰. Depending upon the number of choices in thefirst stage, one will divide up the particles accordingly into as manycontainers. One can use microtiter well plates, individual containers,columns, gels, Terasaki plates, flasks, Merrifield synthesis vessels,etc. The particles will usually be divided up into groups of at leastone particle each, usually a plurality of particles, generally 1000 ormore, and may be 10⁵ or more depending on the total number of particlesand choices involved in the stage.

One would then add the appropriate agents to each of the individualcontainers to process them in stages and add the identifiers whichencode the reagent and stage. Each stage would provide the desiredreaction. Once the reaction(s) is complete, one may wish to wash thebeads free of any reagent, followed by combining all of the beads into asingle mixture and then separating the beads according to the number ofchoices for the next stage. This procedure of dividing beads, followedby the tagging and synthesis stages (or vice versa), and thenrecombining beads is iterated until the combinatorial synthesis iscompleted.

In some instances, the same reaction may be carried out in 2 or morecontainers to enhance the proportion of product having a particularreaction at a particular stage as compared to the other choices. Inother instances, one or more of the stages may involve a portion of thebeads being set aside and undergoing no reaction, so as to enhance thevariability associated with the final product.

In other situations, batches may be taken along different syntheticpathways.

In order to record or encode the synthesis history on the beads, in oneembodiment C or C' or both may be present and subsequent attachment of Cexcludes the presence of C' at each stage one would tag the beadsassociated with each choice and stage with their own unique combinationof identifiers. Alternately one may use a single tag to record or encodethis synthesis history. Depending on the chemistries involved, thistagging may be done prior to, after, or concomitantly with the reactionswhich comprise each choice. Further, as a control, sample beads may bepicked at any stage and a portion of their tags cleaved off and decodedto verify that the correct tags are bound to the sample beads.

As indicated previously, in some instances, portions of the particleswill be segregated into subsets, where each of the subsets would thenundergo a different reaction series. At any time, the portions may berecombined into a single mixture for subsequent reaction. For example,if at one stage one introduces unsaturation, one could provide twosubsets, where in one subset the unsaturation is reduced, while in theother subset the unsaturation is epoxidized. These two subsets couldthen be subjected to different reaction series.

After synthesis of the products is complete, they are screened for adesired property either after detachment of the ligand from the bead orwhile still attached. In the latter case, beads, for example, may beincubated in aqueous buffer with mouse monoclonal antibody Y. Afterincubation and washing, the beads are incubated with alkalinephosphatase-conjugated rabbit (or goat) polyclonal antibody directedagainst mouse antibodies. Using a fluorescent precipitation developingreagent, fluorescent beads with attached monoclonal antibody areidentified and manually separated from the majority of clear, unstainedbeads. Alternatively, the fluorescent beads can be separated using afluorescence-activated cell sorter, so long as the tags are retained onthe bead under the conditions of sorting. Each selected fluorescent beadis subjected to a means for releasing at least some of the tags from thebead.

In instances where the synthesis does not involve the stagewise additionof like units, or where reaction byproducts are formed, there may beinstances where there will be a plurality of compounds on a single beador the structure of the active compound cannot be known from itsreaction history. In accordance with the subject invention, by knowingthe reaction history, one may repeat the synthesis on a larger scale soas to obtain a sufficient amount of the product(s) to isolate theproduct(s) and structurally identify the active compound.

The subject methodology may be illustrated using various reactionsequences. For example, barbiturates may be prepared by combining analdehyde or ketone with an acetate ester to prepare a crotonate underClaisen conditions to provide an unsubstituted to tetrasubstitutedcrotonate. The crotonate may then be combined with a second acetateunder Michael conditions, whereby a glutarate may be obtained having upto 6 substituents. The glutarate may then be combined with ammonia ormonosubstituted amine to provide the barbiturate. By varying thealdehydes and ketones, the acetates and the amines, a great variety ofbarbiturates may be obtained. Where functionalities are present on oneor more of the substituents, such as amino, carboxy, hydroxy, thiol, andthe like, these groups may be protected or modified as desired.

In another example described by Bunin and Ellman, J. Am. Chem. Soc.,114, 10997 (1992), benzodiazepines are produced. One begins thesynthesis with different amino protected substituted2-aminobenzophenones bound to individual particles through, for examplea 4'-oxy group. To each different group of particles in differentvessels, after deprotection, are added a different Fmoc-protectedα-amino acid, either naturally occurring or synthetic, under conditionswhere a peptide bond is formed. After deprotection, internal cyclizationis caused, followed by alkylation on nitrogen with an alkylating agent.In only three stages, a very large number of benzodiazepines may beprepared and the libraries screened for tranquilizing or other activity.

A wide variety of drug analogs may be produced, such as analogs ofantihypertensive agents, e.g. enalapril; β-blockers, e.g. propanolol;antiulcer drugs (H₂ -receptor antagonists) e.g. cimetidine andranitidine; antifungal agents (cholesterol-demethylase inhibitors) e.g.isoconazole; anxiolytics, e.g. diazepam; analgesics, e.g. aspirin,phenacetamide, and fentanyl; antibiotics, e.g. vancomycin, penicillinand cephalosporin; antiinflammatories, e.g. cortisone; contraceptives,e.g. progestins; abortifacients, e.g. RU-456; antihistamines, e.g.chlorphenamine; antitussives, e.g. codeine; sedatives, e.g. barbitol;etc.

An illustrative synthesis of cimetidine analogs could involvehydroxymethylsubstituted histidines, and related heterocycles, where theremaining carbon atoms or nitrogen atoms could be further substituted orunsubstituted, α,ω-aminoalkylthiols, and substituted thioamidine esters,where the groups on nitrogen could be varied, such as nitro, cyano,hydroxy, alkyl, combinations thereof, and the like.

Identifier

The identifiers of this invention may be represented by the Formula I:

    F.sup.1 -F.sup.2 -C-E-C'                                   I

where F¹ -F² ' is a linker which allows for attachment to a support anddetachment of the tag from a support; and

C-E-C' is the tag which is capable of detection and distinguishability;

E is a tag component which (a) allows for detection, such as anelectrophoric group which can be analyzed by gas chromatography or massspectroscopy or (b) allows for detection and for separation;

C and C' are tag components which allow for individually distinguishingone tag from all other tags, usually allowing for separation as a resultof variable length or substitution, for example, varying thechromatographic retention time or the mass spectroscopy ratio m/e;

F² is a linking component capable of being selectively cleaved torelease the tag component; and

F¹ is a functional group which provided for attachment to the support;or

F² is a bond when F¹ is a cleavable group such as OH or carboxy.

Although the identifiers of Formula I are typically added at eachappropriate stage and choice during the combinatorial synthesis, theportion E can be added at the end of the syntheses either before orafter cleavage (preferably photochemically or oxidatively) from thesubstrate. Specifically, where C contains OH, NHR⁴, or SH, E can beattached to C prior to cleavage. Alternatively, if E is attached aftercleavage, the point of attachment at C may be where F² was attached.This is exemplified in the scheme on the following page: ##STR23## whereS=substrate and

n=1-40

Attachment of the identifier to the substrate can be represented asfollows:

    F.sup.1 -F.sup.2 -C-E-C'+S→S-F.sup.1 '-F.sup.2 -C-E-C'

where F¹ '-F² -C-E-C' represents the identifier residue attached to thesubstrate. For example, when the bead is functionalized with anaminomethyl group and F¹ is CO₂ H, then F¹ ' is --C(O)--; when the beadcontains an unsaturated bond and F¹ is N₂ CH--C(O)--, then F¹ ' is═CH--C(O)-- or --CH₂ --C (O)--.

Of particular interest for use as identifiers are compounds of Formula Iof the Formula Ia:

    F.sup.1 -F.sup.2 -(C(E-C').sub.a).sub.b                    Ia

wherein:

F¹ is CO₂ H, CH₂ X, NR¹ R¹, C(O)R¹, OH, CHN₂, SH, C(O)CHN₂, S(O₂)Cl,S(O₂)CHN₂, N₃, NO₂, NO, S(O₂)N₃, OC(O)X, C(O)X, NCO, or NCS;

F² is ##STR24## A is --O, --OC(O)O--, --OC(O)--, or --NHC(O)--; C is abond, C₁ -C₂₀ alkylene optionally substituted by 1-40 F, Cl, Br, C₁ -C₆alkoxy, NR⁴ R⁴, OR⁴, or NR⁴, or --[(C(R⁴)₂)_(m) --Y--Z--Y--(C(R⁴)₂)_(a)Y--Z--Y]_(p) --; with the proviso that the maximum number of carbonatoms in C+C' is preferably 20;

C' is H; F; Cl; C₁ -C₂₀ alkylene optionally substituted by 1-40 F, Cl,Br, C₁ -C₆ alkoxy, NR⁴ R⁴, OR⁴, or NR⁴, or --[(C(R⁴)₂)_(m)--Y--Z--Y--(C(R⁴)₂)_(a) Y--Z--Y]_(p) --;

E is C₁ -C₁₀ alkyl substituted by 1-20 F, Cl or Br; or Q-aryl whereinthe aryl is substituted by 1-7 F, Cl, NO₂, SO₂ R⁵, or substituted phenylwherein the substituent is 1-5 F, Cl, NO₂, or SO₂ R⁵ ;

E-C' may be --H, --OH, or amino;

R¹ is H or C₁ -C₆ alkyl;

R³ is C═O, C(O)O, C(O)NR¹, S, SO, or SO₂ ;

R⁴ is H or C₁ -C₆ alkyl;

R⁵ is C₁ -C₆ alkyl;

a is 1-5;

b is 1-3;

m and n is each 0-20;

p is 1-7;

Q is a bond, O, S, NR⁴, C═O, --C(O)NR⁵, --NR⁵ C(O)--, --C(O)O--, or--OC(O)--;

X is a leaving group such as Br, Cl, triflate, mesylate, tosylate, orOC(O)OR⁵ ;

Y is a bond, O, S, or NR⁴ ;

Z is a bond; phenylene optionally substituted by 1-4 F, Cl, Br, C₁ -C₆alkyl, C₁ -C₆ alkoxy, C₁ -C₆ alkyl substituted by 1-13 F, Cl, or C₁ -C₆alkyloxy substituted by 1-13 F, Cl, or Br; (C(R⁴)₂)₁₋₂₀ ; or (CF₂)₁₋₂₀ ;with the proviso that when Z is a bond one of its adjacent Y's is also abond; and

aryl is a mono- or bi-cyclic aromatic ring containing up to 10 carbonatoms and up to 2 heteroatoms selected from O, S, and N.

In the definitions of F² in Formula Ia, the left-hand bond as depictedattaches to F¹.

Also useful as identifiers are compounds of the Formula Ia';

    F.sup.1 -(C(E-C').sub.a).sub.b                             Ia'

wherein:

F¹ is OH or COOH; and

the remaining definitions are as in Formula Ia.

Preferred compounds of Formula Ia are those wherein;

F¹ is

    CO.sub.2 H, OH, CHN.sub.2, C(O)CHN.sub.2, C(O)X, NCS, or CH.sub.2 X:

F² is ##STR25## C and C' is each independently C₁ -C₂₀ alkyleneunsubstituted or substituted by 1-40 F or Cl, or [O-(CH₂)₂₋₃ ]_(p) ;

E is C₁ -C₁₀ alkyl substituted by 1-20 F or Cl; Q-aryl where aryl is abi-cyclic aromatic ring substituted by 1-7 F or Cl; or Q-phenylsubstituted by 1-5 F, Cl, NO₂, or SO₂ R⁵ ; and

Q is a bond, O, --NR⁵ C(O)--, or --OC(O)--.

Preferred compounds of Formula Ia are those wherein

--C(E-C')_(a) is represented by --(CH₂)₃₋₁₅ --(CF₂)₁₋₁₅ F, --(CH₂)₃₋₁₅-(CCl₂)₁₋₁₅ Cl, --(CH₂ CH₂ --O)₁₋₅ --Ar, --(CH₂ CH₂ CH₂ O)₁₋₅ --Ar or--(CH₂)₁₋₁₂ --O--Ar;

wherein

Ar is pentafluoro- pentachloro-, or pentabromophenyl,2,3,5,6-tetrafluoro-4(2,3,4,5,6-pentafluorophenyl)phenyl,2,4,6-trichlorophenyl, 2,4,5-trichlorophenyl,2,6-dichloro-4-fluorophenyl, or 2,3,5,6-tetrafluorophenyl.

Other preferred compounds of Formula Ia are represented by the formulae:##STR26## wherein Ar is pentafluoro- pentachloro-, or pentabromophenyl,2,3,5,6-tetrafluoro-4(2,3,4,5,6-pentafluorophenyl)phenyl,2,4,6-trichlorophenyl, 2,4,5-trichlorophenyl,2,6-dichloro-4-fluorophenyl, or 2,3,5,6-tetrafluorophenyl.

Other preferred compounds of Formula Ia are those wherein E-C' is H, OH,or NH₂. Such compounds are particularly useful for reaction with an E atthe end of the combinatorial synthesis, especially with an E detectableby fluorescence or electron capture, such as dansyl chloride orpolyhalobenzoylhalide.

The compounds of Formula I can be prepared according to the followingexemplary schemes or other means known to those skilled in the art.##STR27##

The identifier may comprise one or a plurality of identical tags. Theidentifiers will be individual chemical compound(s) which may bedistinguished one from the other and will uniquely identify differentchoices and stages. In this manner, very large combinatorial librariesmay be prepared with a relatively small number of identifiers, usuallyfewer than 50 tags.

During each stage, a combination of identifiers will be added, whichdefines the stage and choice. Each identifier will be bound, eithercovalently or non-covalently to the bead or to the product, usually thebead. Combinations of identifiers are used to provide a binary or othercode at each stage, whereby the choice and stage may be defined. Thecombination of identifiers may include zero or only one identifier.

Tags

So far as the tags (C-E-C') are concerned, the tags which are employedwill be characterized as follows: by being removable from the bead bymeans depending on F², preferably by photolysis or oxidation; by beingindividually differentiable, usually separable; by being stable underthe synthetic conditions; by encoding both stage and choice so as touniquely define the choice of agent used at each stage in the synthesis;desirably, there should be an easy way to identify the various tags withreadily-available equipment which does not require sophisticatedtechnical capabilities to operate; they should be relatively economicaland provide a strong signal based on a relatively few molecules; and thetags should provide sufficient sensitivity to permit distinguishing thetags from the other components which may be present during the tagdeterminations.

The tags may be structurally related or unrelated, as in a homologousseries, repetitive functional groups, related members of the PeriodicChart, different isotopes, combinations thereof, or the like. The tagsmay be used as elements of a binary code, so that one tag can define twochoices, two tags can define four choices, three tags can define eightchoices, five tags can define thirty-two choices, etc. Thus, at eachstage of the synthesis, a relatively small number of tags can designatea much larger number of choices. The tags comprising the identifiers foreach stage may or may not be related to other stages. Each tag for anycombinatorial synthesis must allow for being distinguished from allother tags. In this manner, very large combinatorial libraries may beprepared with a relatively small number of tags, usually fewer than 60tags, more usually fewer than about 50 tags. For each bead, there willusually be at least 0.01 femtomol, more usually 0.001-50 pmol, of eachtag, although lesser or greater amounts may be used in specialcircumstances. The amount of product may also be at least in the samerange and up to at least 10⁴ or more greater, usually being at least0.01 pmol, more usually at least 1.0 pmol and generally not more thanabout 10 nmol. Depending upon the number of beads, the number of stagesand the number of choices per stage, the number of products producedwill usually exceed 10², more usually 10³, and may exceed 10¹⁰, usuallynot exceeding about 10⁸, preferably being in the range of about 10⁴ to10⁸, more usually 10⁵ to 10⁸.

The tags will, for the most part, be organic molecules. Each tag willusually have fewer than about 100 atoms, more usually fewer than about80 atoms, generally fewer than about 60 atoms, other than hydrogen,excluding a linking moiety which would not be retained on release of thetag from the bead. The linking moiety may be of any size, usually beingfewer than about 30 atoms, more usually fewer than 20 atoms, other thanhydrogen. The size of the linking moiety is not critical, but one ofconvenience. The tags may form families of compounds, where all of thecompounds are of a similar nature or may be combinations of differentfamilies, where the compounds may be aliphatic, alicyclic, aromatic,heterocyclic, or combinations thereof. Distinguishing features may bethe number of repetitive units, such as methylene groups in an alkylmoiety, alkyleneoxy groups in a polyalkyleneoxy moiety, halo groups in apolyhalocompound, α- and/or β-substituted ethylenes, where thesubstituents may involve alkyl groups, oxy, carboxy, amino, halo, or thelike; isotopes; etc.

Tag Analysis

Tags may be removed from the bead using reductive, oxidative,thermolytic, hydrolytic, or photolytic conditions depending on thenature of the group F² ; for example, by oxidation of a catechol etherwith ceric ammonium nitrate or by photolysis of a nitrobenzyl ether orester or amide, or by other methods, e.g. as shown in Table 1.

Differentiation of tags can be achieved with physical differences, e.g.molecular weight of the tags or the chromatographic retention time usinggas or liquid chromatography. Positional isomers may have differentretention times. If positional isomers or steroisomers are inadequatefor physical separation, then one could use varying numbers ofsubstituents, e.g. halogens, such as fluorines, methyl groups, oxygroups, or other side chains in conjunction with differing numbers ofunits, e.g. methylene groups or ethyleneoxy groups, to provide thedesired separation. Ratios of radioisotopes could be used, where theradioisotopes provide for differential emission, for example ¹⁴ C and ³H. The physical differences, particularly mass number, can provideinformation about choice and stage. Instead of ¹⁴ C/³ H ratios, onecould use combinations of non-radioactive isotopes, e.g. --CH_(m) D_(a),where m is 0 and up to 3 and n is 3 minus m. For example, by detectingthe varying amounts of up to four different methyl groups using massspectroscopy, one could define a large number of choices.

When E is a bond and C' is H, the tags obtained upon release from thesupport have an active functionality for reaction with a labelingreagent which introduces a detectable tag component E. Conveniently, thefunctionality could be a double bond, particularly an activated doublebond, hydroxy, thio, amino, carboxy, etc. The tag would then be reactedwith an excess of the labeling reagent to provide the product (E-C) foranalysis. In this way a wide variety of labeling reagents could be usedas part of the identifying system, which may not be compatible with thesynthetic strategy for the product of interest. Labeling reagents whichmay be used for detection include haloaromatics (e.g., perfluorobenzylbromide), fluorescers (e.g., dansyl chloride), radioisotopes,chemiluminescers, etc.

While exemplary tags and reactions have been given, it should beunderstood that many other combinations could be employed.

Depending on the chemical and physical nature of the tags, anappropriate method for separation is chosen, desirably one of variouschromatographic procedures including gas chromatography (GC), liquidchromatography (LC) particularly high-performance liquid chromatography(HPLC), thin layer chromatography (TLC), electrophoresis, etc. Insteadof chromatographic procedure, mass spectrometry may be employed forseparation by mass number. Tags include:

for GC: chemically inert organic molecules having different molecularweights including alkanes, alkenes, arenes, halocarbons, ethers,alcohols, silanes, thioethers, etc., particularly halogenated compounds,with or without other functionalities, for electron capture detection ormass spectroscopy detection (MS) with capillary GC separation, and forcompound with elements not normally found in organic chemistry (e.g.,Sn, Ge) for atom emission detection with GC capillary seperation;

for LC, HPLC or TLC: see above for GC, conveniently linear ethers orhydrocarbons with substitution by radioisotopes or combinations ofradioisotopes for radioassay detection or suitable groups forfluorescence detection after separation;

for electrophoresis: see above, particularly functionalized chargedmolecules, e.g. cationic or anionic, particularly organic or inorganicacid groups, where the molecule may be further modified by having adetectable radioisotope or fluorescer for detection in theelectrophoresis;

for mass spectroscopy: see above, particularly different mass numbersdue to different isotopes, different numbers of the same functionalityor different functionalities, different members of a homologous seriesor combinations thereof.

The separation of tags from one another may involve individualtechniques or combinations of techniques, e.g. chromatography andelectrophoresis; gas chromatography and mass spectroscopy; etc.

The tags of the present invention will have a property which allowsdetection at very low levels, usually not greater than nanomol,preferably picomol or less, more preferably femtomol or less, in thepresence of other compounds which may be present at significantly higherlevels. For this reason, specific atomic substitutions may be used torender the labels easily detectable. Such substitutions include:

(a) substitution by electronegative elements, e.g. fluorine or chlorine,for electron capture detection in conjunction with capillary GC ornegative ion mass spectroscopy detection;

(b) substitution by an uncommon element (excluding C, H, and O) foratomic emission detection in conjunction with capillary GC;

(c) substitution by several uncommon elements for atomic emissiondetection to determine the ratio between the elements;

(d) substitution by a radioactive element, e.g. ³ H, for detection byautoradiography or scintillation counting in conjunction with LC, TLC orelectrophoresis;

(e) substitution by a multiplicity of radioactive elements havingdiffering emissions, e.g. ³ H and ¹⁴ C, for detection by autoradiographyor scintillation counting to determine the ratio of the differentradioactive elements.

For single-element substitution (a., b., d. above) a separable mixtureof A tags whose simple presence or absence can be detected would encodeup to 2^(A) different syntheses. For multiple-element substitution (see,c. and e. above) a separable mixture of A tags each having Bdistinguishable states (e.g., different ³ H/¹⁴ C ratios, different Si/Snratios) would be able to encode for up to B^(A) different syntheses.

A wide variety of isotopes exist, where the presence or ratio ofisotopes may provide information as to stage and choice. The isotopesmay be radioactive or non-radioactive. Isotopes of particular interestinclude deuterium, tritium, ¹⁴ C, ³² p, ¹³¹ I, etc.

By employing mixtures of isotopically-modified compounds, one cangreatly expand the information obtained from a single tag compound whichis only distinguished by the presence of isotopes. For example, onecould prepare a mixture of ratios of hydrogen to deuterium, where thevarious ratios could differ by as little as 10% each. By replacinghydrogens with another atom, such as fluorine, one would then have avarying mixture of hydrogens, deuteriums and fluorines, providing for alarge number of different differentiable tags.

Other groups that may be involved could be aromatic rings, which aredifferentially substituted, as to position and functionality. Thus, byhaving substituted benzene rings, where the position of the substitutionand the nature of the substitution can be determined, one can providefor a plurality of molecules which can be distinguished and can providefor both stage and choice information. For example, if C were constantone could detect and discriminate through the substitution pattern on Ewhen E is a polyhalogenated aromatic ring.

There is also the possibility to use fluorescent tags. While fluorescenttags alone may not be sufficient to define a significant number ofstages with a significant number of choices, as referred to above, byproviding for means for separating the fluorescent tagging moleculesbased on variations in C or C', one can individually detect the tags bytheir fluorescence.

The mixture of tags associated with a particular bead may be detachedand subject to an initial separation, where it is desirable to detecteach of the tags separately. Once the group of tags has been separated,each of the tags may then be analyzed based on its particularfunctionalities and distinctive properties. Various techniques which maybe used to detect the particular tags include autoradiography orscintillation counting, electron capture detection, negative or positiveion mass spectroscopy, infrared spectroscopy, ultraviolet spectroscopy,electron spin resonance spectroscopy, fluorescence, and the like.

Another composition may have at least 6 different markers beingassociated in a kit or in a common medium, each marker having adistinguishable moiety which is substantially chemically inert,differing from each other in molecular weight, said markers of theformula:

    Λ-{Δ-(T).sub.α  or (T).sub.α -Δ or Δ.sup.1 -(T).sub.α -Δ.sup.2 }           (1)

where Λ is a linking group which has a functionality for bonding to asolid support and a functionality for detachment from the solid support,which may be included in the functionality for bonding to the solidsupport;

Δ is a distinguishing group, which allows for the distinguishing by aphysical characteristic of each of the markers from each of the othermarkers by other than fluorescence, so as to provide a set of markerswhich allows for coding a multistep synthetic procedure, and includesany remaining functional group after detachment, which was previouslyassociated with the linked group;

Δ¹ and Δ² are portions of the distinguishing group, which togetherdefine a distinguishing group and when joined together come within thedefinition of Δ;

T is a detectable group, which when attached to the distinguishing groupallows for the low level detection of the marker, where the detectablegroup may be present on the marker in the kit or may be added later tothe distinguishing group and if attached to the linking group, includesany remaining functional group after detachment, which was previouslyassociated with the linking group; and α is 0 or 1, indicating that thedetectable group may or may not be present;

    ss-(Λ'-{Δ-(T).sub.α or (T).sub.α -Δ or Δ.sup.1 -(T).sub.α -Δ.sup.2 }).sub.β(2)

where all of the symbols have been defined previously, except asfollows: ss is a solid support; Δ' is a linking group covalently bondedto ss; and β is an integer as to each solid support and is at least sixand usually not more than about 30;

    Λ"-{Δ"-(T).sub.60  or (T").sub.α -Δ or Δ'.sup.1 -(T).sub.α -Δ.sup.2 }          (3)

where all of the symbols have been defined previously, except asfollows: Δ" is hydrogen or the residue of the linking group afterphotolytic cleavage, elimination or other chemical reaction whichresults in detachment from the solid support; Δ" or Δ'¹ is Δ or Δ¹ or amodified Δ or Δ¹, respectively, as a result of the detachment of themarker from the solid support; T" is T or a modified T as a result ofthe detachment of the marker from the solid support;

    T.sub.α -Λ'"{Δ"-(T).sub.α or (T").sub.α -Δ or Δ'.sup.1 -(T).sub.α -Δ.sup.2 }(4)

where all of the symbols have been defined previously and Λ'" is a bondor the remaining portion of the linker group after attachment to T; withthe additional limitation that only one a is 1.

Assays

To determine the characteristic of interest of the product, a widevariety of assays and techniques may be employed.

Frequently, in screening the beads, one will use either single beads ormixtures of beads and determine whether the bead or mixtures showactivity. Thus, the mixtures may involve 10, 100, 1000 or more beads. Inthis way, large groups of compounds may be rapidly screened andsegregated into smaller groups of compounds.

One technique is where one is interested in binding to a particularbiomolecule such as a receptor. The receptor may be a single molecule, amolecule associated with a microsome or cell, or the like. Where agonistactivity is of interest, one may wish to use an intact organism or cell,where the response to the binding of the subject product may bemeasured. In some instances, it may be desirable to detach the productfrom the bead, particularly where physiological activity by transductionof a signal is of interest. Various devices are available for detectingcellular response, such as a microphysiometer, available from MolecularDevices, Redwood City, Calif. Where binding is of interest, one may usea labeled receptor, where the label is a fluorescer, enzyme,radioisotope, or the like, where one can detect the binding of thereceptor to the bead. Alternatively, one may provide for an antibody tothe receptor, where the antibody is labeled, which may allow foramplification of the signal and avoid changing the receptor of interest,which might affect its binding tot he product of interest. Binding mayalso be determined by displacement of a ligand bound to the receptor,where the ligand is labeled with a detectable label.

In some instances, one may be able to carry out a two-stage screen,whereby one first uses binding as an initial screen, followed bybiological activity with a viable cell in a second screen. By employingrecombinant techniques, one can greatly vary the genetic capability ofcells. One can then produce exogenous genes or exogenous transcriptionalregulatory sequences, so that binding to a surface membrane protein willresult in an observable signal, e.g. an intracellular signal. Forexample, one may introduce a leuco dye into the cell, where an enzymewhich transforms the leuco dye to a colored product, particularly afluorescent product, becomes expressed upon appropriate binding to asurface membrane, e.g. β-galactosidase and digalactosidylfluorescein. Inthis manner, by associating a particular cell or cells with a particularparticle, the fluorescent nature of the cell may be determined using aFACS, so that particles carrying active compounds may be identified.Various techniques may be employed to ensure that the particle remainsbound to the cell, even where the product is released from the particle.For example, one may use antibodies on the particle to a surfacemembrane protein, one may link avidin to the surface of the cell andhave biotin present on the particle, etc.

Assays may be performed stagewise using individual particles or groupsof particles or combinations thereof. For example, after carrying outthe combinatorial syntheses, groups of about 50 to 10,000 particles maybe segregated in separate vessels. In each vessel, as to each particle aportion of the product bound to the particle is released. The fractionalrelease may be as a result of differential linking of the product to theparticle or using a limited amount of a reagent, condition or the like,so that the average number of product molecules released per particle isless than the total number of product molecules per particle. One wouldthen have a mixture of products in a small volume. The mixture couldthen be used in an assay for binding, where the binding event could beinhibition of a known binding ligand binding to a receptor, activationor inhibition of a metabolic process of a cell, or the like. Variousassay conditions may be used for the detection of binding activity aswill be described subsequently. Once a group is shown to be active, theindividual particles may then be screened, by the same or a differentassay. One could of course, have a three- or four-stage procedure, wherelarge groups are divided up into smaller groups, etc. and finally singleparticles are screened. In each case, portions of the products on theparticles would be released and the resulting mixture used in anappropriate assay. The assays could be the same or different, the moresophisticated and time consuming assays being used in the later or laststage.

One may also provide for spatial arrays, where the particles may bedistributed over a honeycomb plate, with each well in the honeycombhaving 0 or 1 particle.

The subject methodology may be used to find chemicals with catalyticproperties, such as hydrolytic activity, e.g. esterase activity. Forthis purpose one might embed beads in a semisolid matrix surrounded bydiffusible test substrates. If the catalytic activity can be detectedlocally by processes that do not disturb the matrix, for example, bychanges in the absorption of light or by detection of fluorescence dueto a cleaved substrate, the beads in the zone of catalytic activity canbe isolated and their labels decoded.

Instead of catalytic activity, compounds with inhibitory or activatingactivity can be developed. Compounds may be sought that inhibit oractivate an enzyme or block a binding reaction. To detect beads thatinhibit an enzyme, which beads have an attached product with thisdesirable property, it is advantageous to be able to release theproducts from the beads, enabling them to diffuse into a semisolidmatrix or onto a filter where this inhibition, activation or blockingcan be observed. The beads that form a visualized or otherwisedetectable zone of inhibition, activation or blocking can then be pickedand the tags decoded. In this case it is necessary that a portion of thesynthesized products be attached to the beads by cleavable linkages,preferably a photolabile linkage, while a portion of the tags remainattached to the bead, releasable after picking by a different means thanbefore.

A dialysis membrane may be employed where a layer of beads is separatedfrom a layer of radiolabeled ligand/receptor pair. The bead layer couldbe irradiated with ultraviolet light and the product released from thebead would diffuse to the pair layer, where the radiolabeled ligandwould be released in proportion to the affinity of the compound for thereceptor. The radiolabeled ligand would diffuse back to the layer ofbeads. Since the radiolabel would be proximal to the bead, beadsassociated with radioemission would be analyzed.

Of particular interest is finding products that have biologicalactivity. In some applications it is desirable to find a product thathas an effect on living cells, such as inhibition of microbial growth,inhibition of viral growth, inhibition of gene expression or activationof gene expression. Screening of the compounds on the beads can bereadily achieved, for example, by embedding the beads in a semisolidmedium and the library of product molecules released from the beads(while the beads are retained) enabling the compounds to diffuse intothe surrounding medium. The effects, such as plaques with a bacteriallawn, can be observed. Zones of growth inhibition or growth activationor effects on gene expression can then be visualized and the beads atthe center of the zone picked and analyzed.

One assay scheme will involve gels where the molecule or system, e.g.cell, to be acted upon may be embedded substantially homogeneously inthe gel. Various gelling agents may be used such as polyacrylamide,agarose, gelatin, etc. The particles may then be spread over the gel soas to have sufficient separation between the particles to allow forindividual detection. If the desired product is to have hydrolyticactivity, a substrate is present in the gel which would provide afluorescent product. One would then screen the gel for fluorescence andmechanically select the particles associated with the fluorescentsignal.

One could have cells embedded in the gel, in effect creating a cellularlawn. The particles would be spread out as indicated above. Of course,one could place a grid over the gel defining areas of one or noparticle. If cytotoxicity were the criterion, one could release theproduct, incubate for a sufficient time, followed by spreading a vitaldye over the gel. Those cells which absorbed the dye or did not absorbthe dye could then be distinguished.

As indicated above, cells can be genetically engineered so as toindicate when a signal has been transduced. There are many receptors forwhich the genes are known whose expression is activated. By inserting anexogenous gene into a site where the gene is under the transcriptionalcontrol of the promoter responsive to such receptor, an enzyme can beproduced which provides a detectable signal, e.g. a fluorescent signal.The particle associated with the fluorescent cell(s) may then beanalyzed for its reaction history.

Libraries and Kits

For convenience, libraries and/or kits may be provided. The librarieswould comprise the particles to which a library of products and tagshave been added so as to allow for screening of the products bound tothe bead or the libraries would comprise the products removed from thebead and grouped singly or in a set of 10 to 100 to 1000 members forscreening. The kits would provide various reagents for use as tags incarrying out the library syntheses. The kits will usually have at least4, usually 5, different compounds in separate containers, more usuallyat least 10, and may comprise at least 10² different separated organiccompounds, usually not more than about 10², more usually not more thanabout 36 different compounds. For binary determinations, the mode ofdetection will usually be common to the compounds associated with theanalysis, so that there may be a common chromophore, a common atom fordetection, etc. Where each of the identifiers is pre-prepared, each willbe characterized by having a distinguishable composition encoding choiceand stage which can be determined by a physical measurement andincluding groups or all of the compounds sharing at least one commonfunctionality.

Alternatively, the kit can provide reactants which can be combined toprovide the various identifiers. In this situation, the kit willcomprise a plurality of separated first functional, frequentlybifunctional, organic compounds, usually four or more, generally one foreach stage of the synthesis, where the functional organic compoundsshare the same functionality and are distinguishable as to at least onedeterminable characteristic. In addition, one would have at least one,usually at least two, second organic compounds capable of reacting witha functionality of the functional organic compounds and capable offorming mixtures which are distinguishable as to the amount of each ofsaid second organic compounds. For example, one could have a glycol,amino acid, or a glycolic acid, where the various bifunctional compoundsare distinguished by the number of fluorine or chlorine atoms present,to define stage, and have an iodomethane, where one iodomethane has noradioisotope, another has ¹⁴ C and another has one or more ³ H. By usingtwo or more of the iodomethanes, one could provide a variety of mixtureswhich could be determined by their radioemissions. Alternatively, onecould have a plurality of second organic compounds, which could be usedin a binary code.

As indicated previously one could react the tags after release with amolecule which allows for detection. In this way the tags could be quitesimple, having the same functionality for linking to the particle as tothe detectable moiety. For example, by being linked to a hydroxycarboxylgroup, an hydroxyl group would be released, which could then beesterified or etherified with the molecule which allows for detection.For example, by using combinations of fluoro- and chloroalkyl groups, inthe binary mode, the number of fluoro and/or chloro groups coulddetermine choice, while the number of carbon atoms would indicate stage.

Groups of compounds of particular interest include linkers joined to asubstituted ortho-nitrobenzyloxy group, indanyloxy or fluorenyloxygroup, or other group which allows for photolytic or other selectivecleavage. The linking group may be an alkylene group of from 2 to 20carbon atoms, polyalkyleneoxy, particularly alkyleneoxy of from 2 to 3carbon atoms, cycloalkyl group of from 4 to 8 carbon atoms, haloalkylgroup, particularly fluoroalkyl of from 2 to 20 carbon atoms, one ormore aromatic rings and the like, where the linker provides for thediscrimination between the various groups, by having different numbersof units and/or substituents.

Individual particles or a plurality of particles could be provided asarticles of commerce, particularly where the particle(s) have shown acharacteristic of interest. Based on the associated tags, the reactionhistory may be decoded. The product may then be produced in a largesynthesis. Where the reaction history unequivocally defines thestructure, the same or analogous reaction series may be used to producethe product in a large batch. Where the reaction history does notunambiguously define the structure, one would repeat the reactionhistory in a large batch and use the resulting product for structuralanalysis. In some instances it may be found that the reaction series ofthe combinatorial chemistry may not be the preferred way to produce theproduct in large amounts.

Thus, an embodiment of this invention is a kit comprising a plurality ofseparated organic compounds, each of the compounds characterized byhaving a distinguishable composition, encoding at least one bit ofdifferent information which can be determined by a physical measurement,and sharing at least one common functionality. A preferred embodiment isa kit comprising at least 4 different functional organic compounds.

More preferred is a kit wherein said functional organic compounds are ofthe formula:

    F.sup.1 -F.sup.2 -C-E-C'                                   I

where F¹ -F² is a linker which allows for attachment to and detachmentfrom a solid particle; and C-E-C' is a tag member which can bedetermined by a physical measurement, especially wherein said functionalorganic compounds differ by the number of methylene groups and/orhalogens, nitrogens or sulfurs present.

Also preferred is a kit wherein the C-E-C' portion is removedphotochemically or a kit wherein the C-E-C' portion is removedoxidatively, hydrolytically, thermolytically, or reductively.

Compounds of this invention may be useful as analgesics and/or for thetreatment of inflammatory disease, especially in the case of theazotricyclics acting as antagonists of the meurokin 1/brandykinreceptor. Members of the benzodiazopine library may be useful as amuscle relaxant and/or tranquilizer and/or as a sedative. Members of the23 million Mixed Amide Library may be of use in the treatment ofhypertension on endothelin antagonists or Raynaud's syndrome.

The following examples are offered by way of illustration and not by waylimitation.

In one embodiment the invention is a composition comprising at least 6different components, each component having a distinguishable moiety.The components may be characterized by each moiety being substantiallychemically stable or inert and having an identifiable characteristicdifferent from each of the other moieties. Each moiety is joined to alinking group having an active functionality capable of forming acovalent bond through a linking group to individually separable solidsurfaces, or joined to a group which is detectable at less than 1nanomole, with a proviso that when the moieties are joined to thelinking group, the components are physically segregated. Preferably, thesolid supports are beads. In one embodiment each component comprisesmolecules of different compounds bound to individual separable solidsurfaces, wherein the molecules on the solid surfaces. Preferably, themoieties of the invention define an homologous series and/or a series ofsubstitutions on a core molecule.

The invention herein is also directed to a compound library comprisingat least one hundred unique solid supports. In this compound libraryeach solid support has (1) an individual compound bound to the solidsupport as a major compound bound to the support; and (2) a plurality oftags e.g. tags incapable of being sequenced, where the tags areindividual tag molecules which are physically distinguishable in beingphysically separable and are substituted so as to be detectable at lessthan about a nanomole or have a functional group for bonding to asubstituent which is detectable at less than about at nanomole.Preferably, in the compound library each solid support has at leastabout 6 tags. In another embodiment, in the compound library the tagsdefine a binary code encoding the synthetic protocol used for thesynthesizing of the compound on the solid support.

This invention also provides a method for determining a syntheticprotocol encoded by separable physically different tags in a series anddefining a binary code. In this method at least two tags are employed todefine each stage of the synthetic protocol, there being at least sixtags. The step of the method comprises separating tags by means of theirphysical differences and detecting the tags. The synthetic protocol isdefined a binary code of different tags.

Compounds of this invention may be useful as analgesics and/or for thetreatment of inflammatory disease, especially in the case of theazatricyclics acting as antagonists of the neurokinin 1/bradykininreceptor. Members of the benzediazepine library may be useful as amuscle relaxant and/or tranquilizer and/or as a sedative. Members of the23.5 Million Mixed Amide Library (Example 6) may be of use in thetreatment of hypertension or Raynaud's syndrome by acting as endothelinantagonists.

EXAMPLE 1 Peptide Library

In order to encode up to 10⁹ different syntheses, one could prepare 30different identifiers which carry individual tags capable of beingseparated one from another by capillary GC. For encoding a smallernumber of syntheses, fewer identifiers would be used. The tags wouldnormally be prepared from commercially-available chemicals as evidencedby the following illustration. ω-Hydroxyalkenes-1, where the number ofmethylene groups would vary from 1 to 5, would be reacted with aniodoperfluoroalkane, where the number of CF₂ groups would be 3, 4, 6, 8,10, and 12. By employing a free-radical catalyst, theiodoperfluorocarbon would add to the double bond, where the iodo groupcould then be reduced with hydrogen and a catalyst or a tin hydride. Inthis manner, 30 different tags could be prepared. The chemical procedureis described by Haszeldine and Steele, J. Chem. Soc. (London), 1199(1953); Brace, J. Fluor. Chem., 20, 313 (1982). The highly fluorinatedtags can be easily detected by electron capture, have different GCretention times, so that they are readily separated by capillary GC, arechemically inert due to their fluorinated, hydrocarbon structure andeach bears a single hydroxyl functional group for direct or indirectattachment to particles.

Before attachment to compound precursors, the tags (referred to asT1-T30) would be activated in a way which is appropriate for thechemical intermediates to be used in the combinatorial synthesis. Byappropriate it is intended that a functionality would be added whichallows for ready attachment by a chemical bond to a compound precursoror to the bead matrix itself. The activation process would be applied toeach of the 30 different tags and allow these tags to be chemicallybound, either directly or indirectly, to intermediates in thecombinatorial compound synthesis. For example, a carboxy derivativecould be used for coupling and upon activation the resulting carboxygroup would bond to the particle.

In the case of a combinatorial synthesis of a peptidic compound or otherstructure made of amide-linked organic fragments, the encoding processcould consist of addition of a carboxylic acid-equipped linker. Forexample, the tag would be coupled to the tert.-butyl ester ofo-nitro-p-carboxybenzyl bromide in the presence of sodium hydride. Theester would then be hydrolyzed in dilute trifluoroacetic acid.

Activated identifiers would be coupled to intermediates at each stage inthe combinatorial compound synthesis. The ortho-nitrobenzyl ether partof the activated identifiers is used to allow photochemical detachmentof the tags after completing the combinatorial synthesis and selectingthe most desirable compounds. The detached tags would then be decodedusing capillary GC with electron capture detection to yield a history ofthe synthetic stages used to prepare the compound selected.

While there is an almost unlimited set of chemical stages and methodswhich could be used to prepare combinatorial libraries of compounds, wewill use coupling of α-amino acids to make a combinatorial library ofpeptides as an example of an application of the encoding methodology. Inthis example, we will describe preparation of a library of pentapeptideshaving all combinations of 16 different amino acids at each of the fiveresidue positions. Such a library would contain 16⁵ members. To uniquelyencode all members of this library, 20 detachable tags (T1-T20) asdescribed above would be required.

To prepare the encoded library, we would begin with a large number(>10⁶) of polymer beads of the type used for Merrifield solid phasesynthesis and functionalized by free amino groups. We would divide thebeads into 16 equal portions and place a portion in each of 16 differentreaction vessels (one vessel for each different α-amino acid to beadded). We would then add a small portion (e.g., 1 mol %) of identifiersto each of the amino acid derivatives (e.g., Fmoc amino acids) to becoupled in the first stage of the combinatorial synthesis. The specificcombination of the tags incorporated into the identifiers added wouldrepresent a simple binary code which identifies the amino acid used inthe first stage of synthesis. The 16 amino acids added would beindicated by numbers 1-16 and any such number could be represented.chemically by combinations of the first four tags (T1-T4). In tables 2and 3, a typical encoding scheme is shown in which the presence orabsence of a tag is indicated by a 1 or a 0, respectively. The letter Tmay represent either the the tag or the identifier incorporating thattag.

                  TABLE 2                                                         ______________________________________                                        A typical encoding scheme.                                                        Amino Acid added in first stage                                                                  T4    T3     T2  T1                                    ______________________________________                                        Number 1 (e.g., glycine                                                                          0     0        0   0                                         Number 2 (e.g., alanine) 0 0 0 1                                              Nwmber 3 (e.g., valine) 0 0 1 0                                               Number 4 (e.g., serine) 0 0 1 1                                               Number 5 (e.g., threonine) 0 1 0 0                                             .                                                                             .                                                                             .                                                                            Number 16 (e.g., tryptophan) 1 1 1 1                                        ______________________________________                                    

We would then carry out a standard dicyclohexyl-carbodiimide (DCC)peptide coupling in each of the 16 vessels using the Fmoc amino acidsadmixed with small amounts of the encoding activated identifiers asindicated above. During the couplings, the amino acids as well as smallamounts (e.g., 1%) of the identifiers would become chemically bound tointermediates attached to the beads.

Next the beads would be thoroughly mixed and again separated into 16portions. Each portion would again be placed in a different reactionvessel. A second amino acid admixed with appropriate new activatedidentifiers (T5-T8) would be added to each vessel and DCC coupling wouldbe carried out as before. The particular mixture of the incorporatedtags (T5-T8) would again represent a simple binary code for the aminoacid added in this, the second stage of the combinatorial synthesis.

                  TABLE 3                                                         ______________________________________                                        A typical encoding scheme.                                                        Amino Acid added in second stage                                                                 T8    T7     T6  T5                                    ______________________________________                                        Number 1 (e.g., glycine                                                                          0     0        0   0                                         Number 2 (e.q., alanine) 0 0 0 1                                              Number 3 (e.q., valine) 0 0 1 0                                               Number 4 (e.g., serine) 0 0 1 1                                               Number 5 (e.g., threonine) 0 1 0 0                                             .                                                                             .                                                                             .                                                                            Number 16 (e.g., tryptophan) 1 1 1 1                                        ______________________________________                                    

After the 16 couplings of stage 2 are complete, the beads would be againmixed and then divided into 16 new portions for the third stage of thesynthesis. For the third stage, T9-T12 would be used to encode the thirdamino acid bound to the beads using the same scheme used for stages 1and 2. After the third couplings, the procedure would be repeated twomore times using the fourth amino acids with T13-T16 and the fifth aminoacids with T17-T20 to give the entire library of 1,048,576 differentpeptides bound to beads.

Although the above beads would be visually indistinguishable, any beadmay be chosen (e.g., by selecting based on the interesting chemical orbiological properties of its bound peptide or other target molecule) andits synthetic history may be learned by detaching and decoding theassociated tags.

The precise method used to detach tags will depend upon the particularlinker used to chemically bind it to intermediates in the combinatorialsynthesis of the target compound. In the example above, theortho-nitrobenzyl carbonate linkages, which are known to be unstable to-300 nm light (Ohtsuka, et al., J. Am. Chem. Soc., 100, 8210 [1978]),would be cleaved by photochemical irradiation of the beads. The tagswould then diffuse from the beads into free solution which would beinjected into a capillary gas chromatograph (GC) equipped with asensitive electron capture detector. Since the order in which the tags(T1-T20) emerged from the GC and their retention times under standardconditions were previously determined, the presence or absence of any ofT1-T20 would be directly determined by the presence or absence of theirpeaks in the GC chromatogram. If 1 and 0 represent the presence andabsence respectively of peaks corresponding to T1-T20, then thechromatogram can be taken as a 20-digit binary number which can uniquelyrepresent each possible synthesis leading to each member of the peptidelibrary. The use of halocarbon tags which are safe, economical anddetectable at subpicomole levels by electron capture detection makesthis capillary GC method a particularly convenient encoding scheme forthe purpose.

As an example of using the encoding scheme for the pentapeptide libraryabove, a particular bead is irradiated with light to detach the tags,the solubilized labels injected into a capillary GC and the followingchromatogram obtained ("Peak" line):

    __________________________________________________________________________    Label                                                                             20                                                                              19                                                                              18                                                                              17                                                                              16                                                                              15                                                                              14                                                                              13                                                                              12                                                                              11                                                                              10                                                                              9 8 7 6 5 4 3 2 1 GC Inject                           Peak |  | | |  |                                                                | |                                                         |   |                                                       |                     Binary 1 1 1 1 0 1 0 0 0 0 1 1 0 0 0 1 0 0 1 0                              Stage                                                                          5------                                                                       4-----                                                                        3------                                                                       2-----                                                                        1-----                                                                         AA Tryptophan Threonine Serine Alanine Valine                               __________________________________________________________________________

The "Label" line diagrams the GC chromatogram where T20-T1 peaks (|) areto be found (note the injection is given on the right and thechromatogram reads from right to left). The "Peak" line represents thepresence of labels (T20-T1) as peaks in the chromatogram. The "Binary"line gives presence (1) or absence (0) of peaks as a binary number. The"Stage" line breaks up the binary number into the five different partsencoding the five different stages in the synthesis. Finally, the "AA"line gives the identity of the amino acid which was added in each stageand was given by the binary code in the "Binary" line above.

EXAMPLE 2 Radio-Labeled Tags

In the next illustration, the tags employed are monomethylethers oflinear alkyl-α,ω-diols. The diol would have N+2 carbon atoms, where Ndesignates the stage. The methyl group would be a radiolabeled reagentwhich would have any of a variety of ³ H/¹⁴ C ratios from 1/1 to m/1,where m is the number of choices. The double radiolabel allows foraccurate quantitation of the tritium present in the tag. By having 10different alkylene groups and 10 different radioactive label ratios,10¹⁰ unique ten-member sets of tags are generated. Tags would beattached by first reacting them with activating agents, e.g. phosgene toform a chloroformate, followed by reaction with the F¹ -F² component. Inthis case, F¹ -F² is the o-nitro-p-carboxy-benzyl alcohol protected asthe t-butyl ester. Each time a synthetic stage is carried out, thede-esterified identifier is added directly to the bead, which hascovalently bonded amine or hydroxyl groups, to form amides or esterswith the acid activated using standard chemistry, e.g., carbodiimidecoupling methodology. At the end of the sequential synthesis, the beadsare then screened with a variety of receptors or enzymes to determine aparticular characteristic. The beads demonstrating the characteristicmay then be isolated, the tags detached and separated by HPLC to give aseries of glycol monomethyl ethers which may then be analyzed forradioactivity by standard radioisotope identification methods. Forexample, if the first and second tags to elute from the HPLC column had³ H/¹⁴ C ratios of 5:1 and 7:1 respectively, then the product whichshowed activity had been synthesized by reagent number 5 in stage 1 andreagent number 7 in stage 2.

EXAMPLE 3 2401 Peptide Library

The identifiers employed were 2-nitro-4-carboxybenzyl, O-arylsubstituted ω-hydroxyalkyl carbonate, where alkyl was of from three to12 carbon atoms and aryl was (A) pentachlorophenyl, (B)2,4,6-trichlorophenyl, or (C) 2,6-dichloro-4-fluorophenyl. The tags aredesignated as NAr, wherein N is the number of methylene groups minus twoand Ar is the aryl group. Thus, tag 2A has a butylene group bonded tothe pentachlorophenyl through oxygen. The subject tags can be easilydetected using electron capture gas chromatography at about 100 fmol.

In the subject analysis, the tagging molecules are arranged in their GCelution order. Thus the tag which is retained the longest on the GCcolumn is designated T1 and is associated with the least significant bitin the binary synthesis code number, the next longest retained tag iscalled T2 representing the next least significant binary bit, and so on.Using an 0.2 mM×20M methylsilicone capillary GC column, eighteenwell-resolved tags were obtained where T1 through T18 corresponded to10A, 9A, 8A, 7A, 6A, 5A, 4A, 3A, 6B, 2A, 5B, 1A, 4B, 3B, 2B, 1B, 2C, andIC, respectively.

An encoded combinatorial library of 2401 peptides was prepared. Thislibrary had the amino acid sequence N-XXXXEEDLGGGG-bead, where thevariable X residues were D, E, I, K, L, Q, or S (single letter code).The 4 glycines served as a spacer between the encoded amino acidsequence and the bead. The combinatorial library included the sequenceH₂ N-KLISEEDL, part of the 10 amino acid epitope which is known to bebound by 9E10, a monoclonal antibody directed against the human C-mycgene product. For encoding this library, three binary bits weresufficient to represent the seven alternative reagents for each stage.The code was as follows: 001=S; 010=I; 011=K; 100=L; 101=Q; 110=E;111=D.

The library was synthesized by first preparing the constant segment ofthe library H₂ NEEDLGGGG-bead on 1.5 g of 50-90μ polystyrene synthesisbeads functionalized with 1.1 meq/g of aminomethyl groups using standardsolid phase methods based on t.-butyl side-chain protection and Fmocmain chain protection (Stewart and Young, "Solid Phase PeptideSynthesis", 2nd edition, Pierce Chemical Co., 1984). After deprotectingthe Fmoc groups with diethylamine, the beads were divided into seven 200mg fractions and each fraction placed in a different Merrifieldsynthesis vessel mounted on a single wrist-action shaker. The beads inthe seven vessels were processed independently as follows (see Table3-1). The letter T in this example refers to the tag or to theidentifier incorporating that tag.

                  TABLE 3-1                                                       ______________________________________                                        Vessel                                                                          No. Step 1 Step 2 Step 3 Step 4                                             ______________________________________                                        1     1% T1      DIC, wash Fmoc(tBu)S, Anh.                                                                         Wash                                      2 1% T2 " FmocI, Anh. "                                                       3 1% T1,T2 " Fmoc(Boc)K, Anh. "                                               4 1% T3 " FmocL, Anh. "                                                       5 1% T1,T3 " Fmoc(trityl)Q, "                                                    Anh.                                                                       6 1% T2,T3 " Fmoc(t-butyl)E, "                                                   Anh.                                                                       7 1% T1,T2,T3 " Fmoc(tBu)D, Anh. "                                          ______________________________________                                    

In accordance with the above procedure a sufficient amount of theidentifiers listed in step 1 were attached via their carboxylic acidsusing diisopropylcarbodiimide to tag about 1% of the free amino groupson each bead in the corresponding vessel. The remaining free aminogroups on each bead were then coupled in step 3 to N-protected aminoacid anhydrides. After washing with methylene chloride, isopropanol, andN,N-dimethylformamide, the beads from the seven vessels were combinedand thoroughly mixed. At this point the library had seven members.

After Fmoc deprotection (diethylamine), the beads were again dividedinto seven vessels and processed as before except that in place of theidentifiers used previously, identifiers representing the second stage(T4-6) were used. By repeating the procedure two more times, usingidentifiers T7-9 and then T10-12 analogously, the entire uniquelyencoded library of 7⁴ =2401 different peptides was prepared using only12 identifiers.

To read the synthesis code from a single selected bead, the bead wasfirst washed four times in a small centrifuge tube with 100 μL portionsof DMF, and then resuspended in 1 μL of DMF in a Pyrex capillary tube.After 2 hrs of photolysis with a Rayonet 350 nm light source, the tagsreleased from the bound identifiers were silylated using about 0.1 μLbis-trimethylsilylacetamide and the solution injected into a HewlettPackard capillary gas chromatograph equipped with an 0.2 mM×20Mmethylsilicone fused silica capillary column and an electron capturedetector. The binary synthesis code of the selected bead was directlydetermined from the chromatogram of the tags which resulted.

EXAMPLE 4 Benzodiazepine Library

A combinatorial benzodiazepine library comprising 30 compounds of theformula VIII ##STR28## wherein: R is CH₃, CH(CH₃)₂, CH₂ CO₂ H, (CH₂)₄NH₂, CH₂ C₆ H₄ OH, or CH₂ C₆ H₅ and

R¹ is H, CH₃, C₂ H₅, CH₂ CH═CH₂, or CH₂ C₆ H₅

is constructed per the following scheme. ##STR29##

The benzodiazepines VIII are constructed on polystyrene beads similarlyto the method of Bunin and Ellman (JACS, 114, 10997-10998 [1992]) exceptthat a photolabile linker is incorporated between the bead and thebenzodiazepine (see steps A, B, and C), thus allowing the benzodiazepineto be removed in step G non-hydrolytically by exposure to U.V. light(350 nm in DMF for 10 minutes to 12 hr). Additionally, binary codes areintroduced in steps D and E which allow for a precise determination ofthe reaction sequence used to introduce each of the 6 R's and 5 R¹ 's.After removal of the tags according to step H and analysis by electroncapture detection following GC separation, the nature of the individualR and R¹ groups is determined.

Steps D, E, and F essentially follow the procedure of Bunin and Ellman,but also include the incorporation of identifiers IXa-c in step D andIXd-f in Step E. The identifiers are all represented by Formula IX,##STR30## wherein: IX_(a) indicates n=6;

IX_(b) indicates n=5;

IX_(c) indicates n=4;

IX_(d) indicates n=3;

IX_(e) indicates n=2; and

IX_(f) indicates n=1.

The codes for each of R and R¹ are as follows:

                  TABLE 4-1                                                       ______________________________________                                               IX        R                                                            ______________________________________                                          a CH.sub.3                                                                    b CH(CH.sub.3).sub.2                                                          a,b CH.sub.2 CO.sub.2 H                                                       c (CH.sub.2).sub.4 NH.sub.2                                                   a,c CH.sub.2 --C.sub.6 H.sub.4 -4-OH                                          b,c CH.sub.2 C.sub.6 H.sub.5                                                ______________________________________                                          IX R.sup.1                                                                  ______________________________________                                          d H                                                                           e CH.sub.3                                                                    d,e C.sub.2 H.sub.5                                                           f CH.sub.2 CH═CH.sub.2                                                    d,f CH.sub.2 C.sub.6 H.sub.5                                                ______________________________________                                    

Step A

To a solution of I (1 equiv) in toluene (conc.=0.5 M) is added the Fmocprotected 2-amino-5-chloro-4'-hydroxy-benzophenone (1.3 eq)anddiethylazaodicarboxylate (1.3 eq) and triphenylphosphine (1.3 eq). Themixture is stirred at room temperature for 24 hr. The solvent is removedin vacuo and the residue triturated with ether and filtered and thesolvent again removed in vacuo. The resultant product II is purified bychromatography on silica gel.

Step B

To a solution of II in DCM (0.2 M) stirring at r.t. is added TFA (3equiv.) and the solution is allowed to stir for 12 hr. The solution isevaporated to dryness in vacuo and the residue dissolved in DCM, washedonce with brine and dried (Na₂ SO₄). Filtration and evaporation of thesolvent affords III.

Step C

1% DVB (divinylbenzene) cross-linked polystyrene beads (50μ)functionalized with aminomethyl groups (1.1 mEq/g) are suspended in DMFin a peptide reaction vessel (Merrifield vessel). III (2 equiv) and HOBt(3 equiv) in DMF are added and the vessel shaken for 10 min. DIC (3 eq)is added and the vessel is shaken until a negative Ninhydrin testindicates completion of the reaction after 12 hr.

The DMF is removed and the resin washed with additional DMF (×5) and DCM(×5) before drying in vacuo.

Step D

The dry resin is divided into 6 reaction vessels and is suspended inDCM. The appropriate combinations of identifiers IX_(a-c) (see Table4-1) are added to the flasks and shaken for 1 hr. The Rh(TFA)₂ catalyst(1 mol %) is added to each flask and shaken for an additional 2 hr. Theflasks are drained and the resin washed with DCM (×5). The resin is thentreated with a solution of TFA in DCM (0.01 M) and shaken for 30 min.and then washed again with DCM (×3) followed by DMF (×2). The resin istreated with a 20% solution of piperidine in DMF and shaken for 30 min.and is then washed with DMF (×3) and DCM (×3).

To each flask is added the appropriate Fmoc-protected amino acylfluoride(3 equiv) (when required side-chain functional groups are protected astert-butyl ester (Asp), tert-butyl ether (Tyr) or tert-butyloxycarbonyl(Lys)) with 2,6-di-tert-butyl-4-methylpyridine (10 equiv) and the flasksshaken overnight or until a negative Ninhydrin test is achieved. Theresin is washed once (DCM) and then the six batches are combined andwashed again (DCM, ×5) before drying in vacuo.

Step E

The dry resin is divided into five reaction vessels and is suspended inDCM. The appropriate combinations of identifiers IX_(d-f) (see Table4-1) are added to the flasks. and shaken for 1 hr. The Rh(TFA)₂ catalyst(1 mol %) is added to each flask and shaken for an additional 2 hr. Theflasks are drained and the resin washed with DCM (×5). The resin in thentreated with a solution of TFA in DCM (0.01 M) and shaken for 30 min.and is then washed with DMF (×3) and DCM (×3).

To each flask is added a solution of 5% acetic acid in DMF and themixtures are heated to 60° C. and shaken overnight. The solvent isdrained and then the resin washed with DMF (×5).

Step F

Each batch of resin is suspended in THF and the flasks are cooled to-78° C. To each flask is added a solution of lithiated5-(phenylmethyl)-2-oxazolidinone (2 equiv) in THF and the mixtures areshaken at -78° C. for 1 hr. The appropriate alkylating agent (Table 4-2)(4 equiv) is then added to each reaction flask followed by a catalyticamount of DMF. The vessels are allowed to warm to ambient temperatureand shaken at this temperature for 5 hrs. The solvent is removed byfiltration and the resin washed with THF (×1) and then dried in vacuo.The batches of resin are then combined and washed with THF (×2) and DCM(×2) and the combined resin is then treated with a 95:5:10 mixture ofTFA:water:dimethylsulphide for 2 hrs to remove the side chain protectinggroups.

                  TABLE 4-2                                                       ______________________________________                                                           ALKYLATING                                                   IDENTIFIER AGENT                                                            ______________________________________                                        e                  H.sub.3 CI                                                   d,e C.sub.2 H.sub.5 Br                                                        f BrCH.sub.2 --CH═CH.sub.2                                                d,f BrCH.sub.2 C.sub.6 H.sub.5                                              ______________________________________                                    

Step G

The resultant benzodiazepine can be cleaved from a bead ofpolystyrene-by suspending the bead in DMF and irradiating with U.V. (350nm) for 12 hrs.

Step H

A bead of interest is placed into a glass capillary tube. Into the tubeis syringed 1 μL of 1M aqueous cerium (IV) ammonium nitrate (CAN)solution, 1 μL of acetonitrile and 2 μL of hexane. The tube is flamesealed and then centrifuged to ensure that the bead is immersed in thereagents. The tube is placed in an ultrasonic bath and sonicated from 1to 10 hrs preferably from 2 to 6 hrs.

The tube is cracked open and ˜1 μL of the upper hexane layer is mixedwith ˜0.2 μL of bis(trimethylsilyl)acetamide (BSA) prior to injectioninto the GC and each tag member determined using electron capturedetection, as exemplified in the following scheme. ##STR31##

EXAMPLE 5 117,649 Peptide Library

An encoded library of 117,649 peptides was prepared. This library hadthe sequence H₂ N-XXXXXXEEDLGGGG-bead, where the variable residue X wasD,E,I,K,L,Q or S. This library was encoded using the 18 tags as definedin Example 3; three binary bits being sufficient to represent the sevenamino acids used in each step. The code was: 001=S; 010=I; 011=K; 100=L;101=Q; 110=E; and 111=D, where 1 indicates the presence and 0 indicatesthe absence of a tag.

The constant segment of the library (H₂ NEEDLGGGG-bead) was synthesizedon 1.5 g of 50-80μ Merrifield polystyrene synthesis beads functionalizedwith 1.1 mEq/g of aminomethyl groups using standard solid phase methodsbased on t-Bu sidechain protection and Fmoc-mainchain protection. Afterdeprotecting the N-terminal Fmoc protecting group with diethylamine, thebeads were divided into seven 200 mg portions, each portion being placedinto a different Merrifield synthesis vessel mounted on a singlewrist-action shaker.

The beads in the seven vessels were processed as in Table 3-1 to attachthe sets of identifiers (T1-T3) and the corresponding amino acid to eachportion except that instead of DIC, i-butylchloroformate was used foractivation.

This procedure first chemically attached small amounts of appropriateidentifiers via their carboxylic acids to the synthesis beads. Thisattachment was achieved by activating the linker carboxyl groups asmixed carbonic anhydrides using isobutylchloroformate, and then addingan amount of activated identifier corresponding to 1% of the free aminogroups attached to the beads. Thus, about 1% of the free amino groupswere terminated for each identifier added. The remaining free aminogroups were then coupled in the usual way with the correspondingprotected amino acids activated as their symmetrical anhydrides.

After washing, the seven portions were combined and the Fmoc protectedamino groups were deprotected by treatment with diethylamine. The beadswere again divided into seven portions and processed as before, exceptthat the appropriate identifiers carrying tags T4, T5, and T6 were addedto the reaction vessels.

The procedure of dividing, labelling, coupling the amino acid combiningand main-chain deprotection was carried out a total of six times usingidentifiers bearing tags T1-T18, affording an encoded peptide library of117,649 different members.

Typical Identifier Preparation

To a solution of 8-bromo-1-octanol (0.91 g, 4.35 mmol) and2,4,6-trichlorophenol (1.03 g, 5.22 mmol) in DMF (5 mL) was added cesiumcarbonate (1.70 g, 5.22 mmol) resulting in the evolution of gas and theprecipitation of a white solid. The reaction was stirred at 80° C. for 2hrs. The mixture was diluted with toluene (50 mL) and poured into aseparatory funnel, washed with 0.5 N NaOH (2×50 mL), 1N HCl (2×50 mL)and water (50 mL) and the organic phase was dried (MgSO₄). Removal ofthe solvent by evaporation gave 1.24 g (87% yield) of tag as a clearoil.

The above tag (0.81 g, 2.5 mmol) was added to a 2 M solution of phosgenein toluene (15 mL) and stirred at room temperature for 1 hr. The excessphosgene and the toluene were removed by evaporation and the resultingcrude chloroformate was dissolved in DCM (5 mL) and pyridine (0.61 mL,7.5 mmol). tert-Butyl 4-hydroxy-methyl-3-nitrobenzoate (Barany andAlbericio, J. Am. Chem. Soc., (1985), 107, 4936-4942) (0.5 g, 1.98 mmol)was added and the reaction mixture stirred at room temperature for 3hrs. The solution was diluted with ethyl acetate (75 mL) and poured intoa separatory funnel. After washing with 1N HCl (3×35 mL), saturatedNaHCO₃ (2×35 mL) and brine (35 mL), the organic phase was dried (MgSO₄).The solvent was removed by evaporation and the residue purified bychromatography on silica gel (5% to 7.5% ethyl acetate in petroleumether) affording 0.95 g (79% yield) of the identifier tert-butyl esteras a clear oil.

Trifluoroacetic acid (3 mL) was added to a solution of the identifiertert-butyl ester (0.95 g, 1.57 mmol) in DCM (30 mL) to deprotect thelinker acid (i.e., F¹ -F² of Formula I) and the solution was stirred atroom temperature for 7 hrs. The mixture was then evaporated to drynessand the residue redissolved in DCM (30 mL). The solution was washed withbrine (20 mL) and the organic phase dried (MgSO₄). Removal of thesolvent by evaporation gave 0.75 g (87% yield) of the identifier (6B) asa pale yellow solid. (Tag nomenclature is the same as in Example 3).

Typical Encoded Library Synthesis Step

Nα-Fmoc-E(tBu)-E(tBu)-D(tBu)-L-G4-NH-resin was-suspended in DMF (20 mL)and shaken for 2 min. After filtering, 1:1 diethylamine:DMF (40 mL) wasadded to remove the Fmoc protecting groups and the resin was shaken for1 hr. The resin was separated by filtration and washed with DMF (2×20mL, 2 min each); 2:1 dioxane: water (2×20 mL, 5 min each), DMF (3×20 mL,2 min each), DCM (3×20 mL, 2 min each) then dried in vacuo at 25° C.(The resin was found to have 0.4 mmol/g amino groups by picric acidtitration at this stage.)

150 mg Portions of the resin were placed into seven Merrifield vesselsand suspended in DCM (5 mL). The appropriate identifiers were activatedas their acyl carbonates as follows (for the first coupling): T1 (6.6mg, 0.0098 mmol) was dissolved in anhydrous ether (2 mL) and pyridine(10 μL) was added. Isobutyl chloroformate (1.3 μL, 0.0096 mmol) wasadded as a solution in anhydrous ether (0.1 mL). The resulting mixturewas stirred at 25° C. for 1 hr. during which time a fine whiteprecipitate formed. The stirring was stopped and the precipitate wasallowed to settle for 30 min. Solutions of the acylcarbonates of T2 andT3 were prepared in the same way. Aliquots (0.25 mL) of the supernatantsolution of activated identifiers were mixed to give the appropriate3-bit binary tag codes and the appropriate coding mixtures ofidentifiers were added to each of the seven synthesis vessels. Thevessels were shaken in the dark for 12 hrs, and then each was washedwith DCM (4×10 mL, 2 min each). A solution of the symmetrical anhydrideof an Nα-Fmoc amino acid in DCM (3 equivalents in 10 mL) was then addedto the corresponding coded batch of resin and shaken for 20 min. 5%N,N-diisopropylethylamine in DCM (1 mL) was added and the mixture shakenuntil the resin gave a negative Kaiser test.

The resin batches were filtered and combined, and then washed with DCM(4×20 mL, 2 min each), isopropanol (2×20 mL, 2 min each), DCM (4×20 mL,2 min each). The next cycle of labelling/coupling was initiated by Fmocdeprotection as described above.

After Fmoc deprotection of the residues in the last position of thepeptide, the side chain functionality was deprotected by suspending theresin in DCM (10 mL), adding thioanisole (2 mL), ethanedithiol (0.5 mL)and tri-fluoroacetic acid (10 mL) then shaking for 1 hr at 25° C. Theresin was then washed with DCM (6×20 mL, 2 min each) and dried.

Electron Capture Gas Chromatography Reading of Code

A single, selected bead was placed in a Pyrex capillary tube and washedwith DMF (5×10 μL). The bead was then suspended in DMF (1 μL) and thecapillary was sealed. The suspended bead was irradiated at 366 nm for 3hrs to release the tag alcohols, and the capillary tube subsequentlyplaced in a sand bath at 90° C. for 2 hrs. The tube was opened andbis-trimethylsilyl acetamide (0.1 mL) was added to trimethylsilylate thetag alcohols. After centrifuging for 2 min., the tag solution above thebead (1 μL) was injected directly into an electron capture detection,capillary gas chromatograph for analysis. Gas chromatography wasperformed using a Hewlett Packard Series II Model 5890 gas chromatographequipped with a 0.2 mm×20 m methylsilicone fused silica capillary columnand an electron capture detector. Photolysis reactions were performedusing a UVP "Black Ray" model UVL 56 hand-held 366 nm lamp.

Antibody Affinity Methods

The anti-C-myc peptide monoclonal antibody 9E10 was prepared fromascites fluid as described in Evans et al., Mol. Cell Biol., 5,3610-3616 (1985) and Munro and Pelham, Cell, 48, 899-907 (1987). To testbeads for binding to 9E10, beads were incubated in TBST [20 mM Tris-HCl(pH 7.5), 500 mM NaCl and 0.05% Tween-20] containing 1% bovine serumalbumin (BSA) to block non-specific protein binding sites. The beadswere then centrifuged, resuspended in a 1:200 dilution of 9E10 ascitesfluid in TBST+1% BSA and incubated overnight at 4° C. Beads weresubsequently washed three times in TBST and incubated for 90 min. atroom temperature in alkaline phosphatase-coupled goat antimouse IgGantibodies (Bio-Rad Laboratories), diluted 1:3000 in TBST+1% BSA. Afterwashing the beads twice in TBST and once in phosphatase buffer (100 mMTris-HCl, pH 9.5, 100 mM NaCl and 5 mM MgCl₂), the beads-were incubated1 hr at room temperature in phosphatase buffer containing oneone-hundreth part each of AP Color Reagents A & B (Bio-RadLaboratories). To stop the reaction, the beads were washed twice in 20mM sodium EDTA, pH 7.4. Solution phase affinities between 9E10 andvarious peptides were determined by a modification of the competitiveELISA assay described by Harlow et al., Antibodies: a Laboratory Manual,570-573, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.

From a 30 mg sample of the combinatorial library of peptides, 40individual beads were identified which stained on exposure to theanti-C-myc monoclonal antibody. Decoding of these positive-reactingbeads established the ligand's reaction sequence as the myc epitope(EQKLISEEDL) or sequences that differed by one or two substituents amongthe three N-terminal residues.

EXAMPLE 6 23,540,625 Mixed Amide Library

The encoding technique was tested further by the preparation of acombinatorial library of 23,540,625 members consisting of peptides andother amide compounds.

The synthesis was carried out using 15 different reagents in 5 steps and31 different reagents in the sixth step. Four identifiers were used toencode each of the 5 steps with 15 reagents and five identifiers wereused in the final step with 31 reagents. A label set of 25 identifierswas therefore prepared. 2-Nitro-4-carboxybenzyl, O-aryl substitutedω-hydroxyalkyl carbonate identifiers were employed, where the tagcomponents were comprised of an alkyl moiety of from 3 to 12 carbonatoms and the aryl moieties were (A) pentachlorophenyl, (B)2,4,5-trichlorophenyl, (C) 2,4,6-trichlorophenyl, or (D)2,6-dichloro-4-fluorophenyl. A set of 25 tags was prepared usingappropriate alkyl chains lengths with A, B, C or D, separable using a0.2 mM×25M methylsilicone GC column. The chemical compositions of tagsT1-T25 (where T1 represents the tag with the longest retention time, andT25 the tag with the shortest retention time) are summarized below:

    ______________________________________                                        T1   10A     T6     10C  T11  7B   T16  5C   T21  2B                            T2 9A T7 9B T12 7C T17 4B T22 2C                                              T3 8A T8 9C T13 6B T1B 4C T23 1B                                              T4 7A T9 8B T14 6C T19 3B T24 1C                                              T5 10B T10 8C T15 5B T20 3C T25 2D                                          ______________________________________                                    

The designations 10A, 9A, etc. are as described in Example 3.

The fifteen reagents used in the first five stages and the codeidentifying them are represented below where 1 represents the presenceof tag and 0 the absence thereof.

    ______________________________________                                        REAGENT         CODE                                                          ______________________________________                                        L-serine        (0001)                                                          D-serine (0010)                                                               L-glutamic acid (0011)                                                        D-glutamic acid (0100)                                                        L-glutamine (0101)                                                            D-glutamine (0110)                                                            L-lysine (0111)                                                               D-lysine (1000)                                                               L-Proline (1001)                                                              D-Proline (1010)                                                              L-phenylalanine (1011)                                                        D-phenylalanine (1100)                                                        3-amino-benzoic (1101)                                                        acid                                                                          4-aminophenyl (1110)                                                          acetic acid                                                                   3,5-diamino- (1111)                                                           benzoic acid                                                                ______________________________________                                    

The 31 reagents and the code representing them in the sixth stage arerepresented below:

    ______________________________________                                        REAGENT             CODE                                                      ______________________________________                                        L-serine            (00001)                                                     D-serine (00010)                                                              L-glutamic acid (00011)                                                       D-glutamic acid (00100)                                                       L-glutamine (00101)                                                           D-glutamine (00110)                                                           L-lysine (00111)                                                              D-lysine (01000)                                                              L-proline (01001)                                                             D-proline (01010)                                                             L-phenylalanine (01011)                                                       D-phenylalanine (01100)                                                       3-amino-benzoic acid (01101)                                                  4-aminophenyl acetic acid (01110)                                             3,5-diamino-benzoic acid (01111)                                              Succinic Anhydride (10000)                                                    Tiglic acid (10001)                                                           2-pyrazine carboxylic acid (10010)                                            (±)thioctic acid (10011)                                                   1-piperidinepropionic acid (10100)                                            piperonylic acid (10101)                                                      6-methylnicotinic acid (10110)                                                3-(2-thienyl)acryiic acid (10111)                                             methyl iodide (11000)                                                         tosyl chloride (11001)                                                        p-toluenesulfonyl isocyanate (11010)                                          3-cyanobenzoic acid (11011)                                                   phthallic anhydride (11100)                                                   acetic anhydride (11101)                                                      ethyl chloroformate (11110)                                                   mesylchloride (11111)                                                       ______________________________________                                    

A spacer of six glycine units was prepared on the beads using standardmethods. The variable region was constructed using butyl sidechainprotection, and amino groups were protected as Fmoc derivatives. Amidebonds were formed by activation of the carboxylic acid with DIC andHOBt.

EXAMPLE 7 Hetero-Diels-Alder Library

A combinatorial hetero Diels-Alder library comprising 42 compounds ofthe formula: ##STR32## wherein; R¹ is H, CH₃ O, F₃ C, F₃ CO, H₅ C₆ O, orC₆ H₁₁ ;

R² is H, CH₃, or CH₃ O;

R³ is H (when n=2), or CH₃ (when n=1); and ##STR33## was constructed perthe following scheme: ##STR34##

The azatricyclic products (VI) were constructed on polystyrene beads andwere linked to the beads by a photocleavable linker allowing theazatricycle (VII) to be removed from the bead by exposure to U.V. light(350 nm in DMF). The binary codes introduced in steps C,D and E allow aunique determination of the reaction sequence used to introduce ArR, R¹,R² and R³. The encoding tags were removed according to step G andanalyzed by electron capture detection following GC separation.

The identifiers used in this scheme are represented by the formula X:##STR35## wherein; X_(a) indicates n=10

X_(b) indicates n=9

X_(c) indicates n=8

X_(d) indicates n=7

X_(e) indicates n=6

X_(f) indicates n=5

X_(g) indicates n=4

The codes for each of R, R₁, R², R³ are as follows:

                  TABLE 7-1                                                       ______________________________________                                        ______________________________________                                          a                                                                                                          3  R = H #                                        - b                                                                                                       3  R = Cl                                         - a, b                                                                                                    4  STR38##                                        -                                                                          c        R.sup.1 = H R.sup.2 = H                                                d R.sup.1 = H R.sup.2 = CH.sub.3                                              d, c R.sup.1 = OCH.sub.3 R.sup.2 = OCH.sub.3                                  e R.sup.1 = CF.sub.3 R.sup.2 = H                                              e, c R.sup.1 = C.sub.6 H.sub.5 O R.sup.2 = H                                  e, d R.sup.1 = F.sub.3 CO R.sup.2 = H                                         e, d, c R.sup.1 = C.sub.6 H.sub.11 R.sup.2 = H                                f R.sup.3 = CH.sub.3 n = 1                                                    g R.sup.3 = H n = 2                                                         ______________________________________                                    

Step A

To a solution of I (2.03 g, 8 mmol), 4-hydroxybenzaldehyde (1.17 g, 9.6mmol) and triphenylphosphine (2.73 g, 10.4 mmol) in toluene (20 mL)stirring at 0° C. was added over a period of 30 minutesdiethylazodicarboxylate. The solution was allowed to warm and stirredfor 1 hour once ambient temperature had been reached. The solution wasconcentrated by removal of approximately half of the solvent in vacuoand was then triturated with ether. The mixture was then filtered andthe residue was washed thoroughly with ether. The solvent was removed invacuo and the residue was purified by chromatography on silica gel (15%ethyl acetate in hexane) affording 1.3 g of the ether IIa (47% yield).

2-chloro-4-hydroxybenzaldehyde and 2-hydroxy-1-naphthaldehyde werecoupled to I in analogous fashion affording ethers IIb and c in yieldsof 91% and 67%, respectively.

Step B

To a solution of ether IIa (0.407 g, 1.14 mmol) in DCM (20 mL) stirringat room temperature was added TFA (8 mL).

The solution was allowed to stir for 6 hrs. The solution was evaporatedto dryness in vacuo affording 0.343 g of acid IIIa (100% yield). EthersIIb and IIc were deprotected analogously affording acids IIIb and c inyields of 92% and 100% respectively.

Step C

Into a peptide reaction vessel (Merrifield vessel) were measured 1% DVB(divinylbenzene) cross-linked polystyrene beads (50-80μ) functionalizedwith aminomethyl groups (1.1 meq/g) (200 mg of resin). The resin wassuspended in DMF (2 mL) and shaken for 20 min. The acid IIIa (38 mg, 2equiv.), 1-hydroxybenzotriazole (40 mg, 2 equiv) anddiisopropylcarbodiimide (38 mg, 2 equiv) were added and the mixtureshaken until a negative Ninhydrin test was achieved (22 hr). Thesolution was removed by filtration and the resin was washed with DCM(8×10 mL).

The resin was resuspended in DCM (5 mL), identifier Xa (15 mg) was addedand the flask was shaken for 1 hr. Rh(TFA)₂ catalyst (1 mol %) was addedand the flasks shaken for 2 hrs. The solvent was removed by filtrationand the resin resuspended in DCM (5 mL). Trifluoroacetic acid (1 drop)was added and the vessel shaken for 20 min. The solvent was removed byfiltration, and the resin was washed with DCM (8×10 mL).

In an analogous fashion, acids IIIb and IIIc were attached to the resinand were encoded with the appropriate identifiers, i.e., Xb for acidIIIb and Xa and Xb for acid IIIc. The three batches of resin werecombined, mixed, washed, and dried.

Step D

The dry resin was divided into 7 equal portions (87 mg) which were putinto seven peptide reaction vessels (Merrifield vessels) which werewrapped with heat tape. The resin in each vessel was suspended intoluene (10 mL) and shaken for 20 min. An appropriate amount of oneaniline was then added to each flask (see Table 7-2).

                  TABLE 7-2                                                       ______________________________________                                        FLASK    ANILINE        AMOUNT ADDED                                          ______________________________________                                        1        Aniline        3         mL                                            2 3,5-dimethylaniline 3 mL                                                    3 3,4,5-trimethaxyaniline 2 g                                                 4 4-trifluoromethylaniline 3 mL                                               5 4-phefloxyaniline 2 g                                                       6 4-trifluoromethoxyaniline 3 mL                                              7 4-cyclohexylaniline 2 g                                                   ______________________________________                                    

The heating tape was connected and the reaction mixtures shaken at 70°C. for 18 hrs. The heat tape was disconnected and the solvent wasremoved by filtration and each batch of resin was washed with dry DCM(4×10 mL), ether (10 mL), toluene (10 mL) and DCM (2×10 mL). Each of theportions was then suspended in DCM (5 mL) and to each flask was addedthe appropriate identifier or combination of identifiers (Xc-e)(15mg)(see Table 7-1). The flasks were shaken for 1 hr. and then Rh(TFA)₂(1 mol %) was added to each flask and shaking continued for 2 hrs.

The solvent was then removed and each batch of resin was re-suspended inDCM (5 mL) to which was added TFA (1 drop). This mixture was shaken for20 min., then the solvent was removed by filtration. The batches ofresin were then washed (DCM, 1×10 mL) and combined, washed again withDCM (3×10 mL) and then dried thoroughly in vacuo.

Step E

The dried resin was divided into two equal portions (0.3 g) and each wasplaced in a peptide reaction vessel. The resin batches were washed withDCM (2×10 mL) and then resuspended in DCM (5 mL). To one flask was addedthe identifier Xf (15 mg) and to the other was added Xg (15 mg). Theflasks were shaken for 1 hr. prior to the addition of Rh(TFA)₂ catalyst(1 mol %). The flasks were shaken for 2 hrs. and then the solvent -wasremoved by filtration. Each batch of resin was washed with DCM (3×10mL), and each was then resuspended in DCM (5 mL).

The appropriate enol ether (1 mL) (see Table 7-1) was added to theflasks and the vessels shaken for 30 min. To each flask was added asolution of BF₃.OEt₂ (0.5 mL of a 5% solution in DCM) and the flaskswere shaken for 24 hrs. Removal of the solvent by filtration wasfollowed by washing of the resin with DCM (10 mL) and the resin was thencombined. The beads were then washed further with DCM (5×10 mL), DMF(2×10 mL) methanol (2×10 mL) and DCM (2×10 mL). The resin was then driedthoroughly in vacuo.

Step F

To confirm the identity of the products produced in theHetero-Diels-Alder library one example was completed on a large scale toallow confirmation of the structure by spectroscopic means. Theprocedure followed was essentially the same method as described for thecombinatorial library. In step A 4-hydroxybenzaldehyde was coupled tothe photolabile group. In step D, aniline was condensed with thealdehyde. In step E, the enol ether was formed with4,5-dihydro-2-methylfuran.

The photolysis of the compound (step F) was performed by suspending 100mg of the beads in DMF (0.3 mL) and irradiating the beads with UVP"Black Ray" model UVL 56 hand-held 366 nm lamp for 16 hrs. The DMF wasremoved to one side by pipette and the beads rinsed with additional DMF(2×3 mL). The original solution and the washings were combined and thesolvent removed in vacuo. NMR analysis of the reaction mixture showed itto contain the desired azatricycle by comparison to the authenticsample.

Step G

A bead of interest was placed into a pyrex glass capillary tube sealedat one end. A solution (1 μL) of 1M aqueous cerium (IV) ammonium nitrateand acetonitrile (1:1) was syringed into the tube, and the tube was thencentrifuged so that the bead lay on the bottom of the capillary and wascompletely immersed by the reagent solution. Hexane (2 μL) was added bysyringe and the tube was again centrifuged. The open end of thecapillary was flame-sealed and placed in an ultrasonic bath for 4 hrs.The capillary was then placed inverted into a centrifuge and spun suchthat the aqueous layer was forced through the hexane layer to the bottomof the tube. This extraction process was repeated 3 or 4 times and thetube was then opened. The hexane layer (1.5 μL) was removed by syringeand placed into a different capillary containing BSA (0.2 μL). This tubewas sealed and centrifuged until the reagents were thoroughly mixed. Aportion of the solution (ca. 1 μL) was removed and injected into a gaschromatography machine with a 25M×0.2 mM methylsilicone fused silicacolumn with electron capture detection for separation and interpretationof the tag molecules.

The sample was injected onto the GC column at 200° C. and 25 psi ofcarrier gas (He₂). After 1 minute the temperature was increased at arate of 20° C. per minute to 320° C., and the pressure was increased ata rate of 2 psi per minute to 40 psi. These conditions are shown in thefollowing diagram: ##STR39## The following results were obtained withfour randomly selected beads:

    ______________________________________                                        Bead 1                                                                              TAG DETECTED                                                                          Xf         Xe Xd Xc                                                                             Xb Xa                                         ______________________________________                                          Ar   2-Hydroxy naphthyl                                                       R.sup.1  C.sub.6 H.sub.11                                                     R.sup.2  H                                                                    R.sup.3 CH.sub.3 (n = 1)                                                    ______________________________________                                        Bead 2                                                                              TAG DETECTED                                                                          Xg       Xe Xd Xc                                                                             Xb                                              ______________________________________                                          Ar   2-chloro-4-hydroxyphenyl                                                 R.sup.1  C.sub.6 H.sub.11                                                     R.sup.2  H                                                                    R.sup.3 H (n = 2)                                                           ______________________________________                                        Bead 3                                                                              TAG DETECTED                                                                          Xg         Xe Xd Xb Xa                                          ______________________________________                                          Ar   2-Hydroxy naphthyl                                                       R.sup.1  F.sub.3 CO                                                           R.sup.2  H                                                                    R.sup.3 H (n = 2)                                                           ______________________________________                                        Bead 4                                                                              TAG DETECTED                                                                          Xf        Xe Xd Xb                                              ______________________________________                                          Ar   2-chloro-4-hydroxyphenyl                                                 R.sup.1  F.sub.3 CO                                                           R.sup.2  H                                                                    R.sup.3 CH.sub.3 (n = 1)                                                    ______________________________________                                    

EXAMPLE 8 Benzodiazepine Library

Following the procedure of Example 4, a combinatorial library isconstructed of the Formula X ##STR40## wherein R is a radical of anaturally occurring D or L amino acid;

R¹ is H, C_(l) -C₆ alkyl, lower alkenyl, C₁ -C₆ alkylamine, carboxy C₁-C₆ alkyl, or phenyl C₁ -C₆ alkyl wherein the phenyl is optionallysubstituted by lower alkyl, F, Cl, Br, OH, NH₂, CO₂ H, or O-lower alkyl;

R² is H or CO₂ H;

R³ is H or OH;

R⁴ is H or Cl;

with the provisos that when R³ is OH, R² is H and when R² is carboxy, R³is H.

This library is released from a plurality of encoded beads of thegeneral formula ##STR41## wherein IX_(n) is a plurality of identifiersof the Formula Ia wherein said plurality represents an encoded scheme;

S is a substrate;

F¹ -F² is the residue of the linker member of Formula Ia; and

R, R¹, R², and R⁴ are as defined for Formula X.

EXAMPLE 9 Typical Identifier Preparations

The diazo compound identifiers which are attached to the resin viacarbene formation are prepared as exemplified.

Compounds of the general formula ##STR42## wherein n is 0-10 and

Ar is pentachlorophenol, 2,4,6-trichlorophenol, 2,4,5-trichlorophenol,or 2,6-dichloro-4-fluorophenol

are prepared as follows.

To a solution of 1-hydroxy-4-(2,6-dichloro-4-fluoro-phenoxy)butane (0.38g, 1.5 mmol), isovanillin (0.228 g, 1.5 mmol) and triphenylphosphine(0.393 g, 1.5 mmol) in THF (8 mL) was added diethylazodicarboxylate(0.287 g, 1.7 mmol). The solution stirred at r.t. for 36 hrs. Thesolvent was removed in vacuo and the residue purified by chromatographyon silica gel (with a mixture of 20% ethyl acetate and 80% petroleumether) affording 0.45 g of the aldehyde (77% yield).

The aldehyde (100 mg, 0.26 mmol) was dissolved in acetone (8 mL) and wastreated with a solution of KMnO₄ (61 mg, 0.39 mmol) in acetone (4 mL)and water (4 mL). The reaction stirred at room temperature for 13 hrs.The mixture was diluted with ethyl acetate (100 mL) and water (50 mL)and the layers were separated. The aqueous layer was extracted withadditional ethyl acetate (2×100 mL). The combined organic layers werewashed with water (50 mL) and dried (MgSO₄). Removal of the solventafforded 109 mg of the benzoic acid (93% yield).

A solution of the acid (76 mg, 0.188 mmol) in methylene chloride (2 mL)was treated with oxalylchloride (36 mg, 0.28 mmol) and catalytic DMF.After stirring for 10 min at room temperature slow but steady evolutionof gas was observed. Stirring continued for 2 hrs. when the solution wasdiluted with DCM (15 mL) and washed with saturated aqueous sodiumhydrogencarbonate solution (5 mL). The layers were separated. Theorganic layer was dried (Na₂ SO₄) and the solvent evaporated affordingthe benzoyl chloride as pale yellow crystals.

The benzoyl chloride was dissolved in methylene chloride (5 mL) and wasadded to a stirring solution of diazomethane in ether at -78° C. Thecold bath was allowed to warm up and the mixture allowed to stir for 5hrs at room temperature. The solvents and excess diazomethane wereremoved in vacuo and the residue purified by chromatography on silicagel using gradient elution method where the concentration of ethylacetate ranged from 10% to 40 % in hexanes affording 48 mg of the diazocompound (60% yield).

Compounds of the general formula: ##STR43## wherein; n is 0-10 and Ar ispentachlorophenol, 2,4,6-trichlorophenol, 2,4,5-trichlorophenol, or2,6-dichloro-4-fluorophenol are prepared as follows.

Methyl vanillate (0.729 g, 4.0 mmole),1-hydroxy-9-(2,3,4,5,6-pentachlorophenoxy)nonane (1.634 g, 4.0 mmole)and triphenylphosphine (1.259 g, 4.8 mmole) were dissolved in 20 mL drytoluene under argon. DEAD (0.76 mL, 0.836 g, 4.8 mmole) was addeddropwise, and the mixture was stirred at 25° C. for one hour. Thesolution was concentrated to half volume and purified by flashchromatography eluting with DCM to give 1.0 g (1.7 mmole, 43%) of theproduct as a white crystalline solid.

The methyl ester above (1.0 g, 1.7 mmole) was dissolved in 50 mL THF, 2mL water was added followed by lithium hydroxide (1.2 g, 50 mmole). Themixture was stirred at 25° C. for one hour then refluxed for five hours.After cooling to 25° C. the mixture was poured onto ethyl acetate (200mL) and the solution was washed with 1 M HCl (50 mL ×3) then sat. aq.NaCl (1×50 mL) and dried over sodium sulfate. The solvent was removedand the crude acid azeotroped once with toluene.

The crude material above was dissolved in 100 mL toluene, 10 mL (1.63 g,14 mmole) thionyl chloride was added, and the mixture was refluxed for90 min. The volume of the solution was reduced to approximately 30 mL bydistillation, then the remaining toluene removed by evaporation. Thecrude acid chloride was dissolved in 20 mL dry DCM and cooled to -78° C.under argon and a solution of approximately 10 mmole diazomethane in 50mL anhydrous ether was added. The mixture was warmed to room temperatureand stirred for 90 min. Argon was bubbled through the solution for 10min. then the solvents were removed by evaporation and the crudematerial was purified by flash chromatography eluting with 10-20% ethylacetate in hexane. The diazoketone (0.85 g, 1.4 mmole, 82% over threesteps) was obtained as a pale yellow solid.

The following identifiers have been prepared as described above:

Photolabile Cleavage

50 Identifiers were prepared of the formula: ##STR44## wherein: Ar is:##STR45## and n is 1,2,3,4,5,6,7,8,9, and 10. Oxidative Cleavage Type I

7 Identifiers were prepared of the formula ##STR46## wherein: Ar is##STR47## and n is 4,5,6,7,8,9, and 10. Oxidative Cleavage Type II

13 Identifiers were prepared of the formula ##STR48## wherein: Ar is##STR49## and n is 1,2,3,4,5,6,7,8,9,10; and wherein:

Ar is ##STR50## and n is 0,3, and 9.

EXAMPLE 10 Encoding Combinatorial Libraries with Tags Readable by MassSpectroscopy

The tags 4, 11 and 13 (Scheme 8) of the same structure, but differentmolecular weights due to varying deuterium substitution, were eachsynthesized (Schemes 9 and 10) and separately analyzed by massspectroscopy (MS). Among MS techniques, positive chemical ionizationmass spectroscopy (PCIMS) gave minimal fragmentation of the tag, suchthat only the molecular ion ([M+NH₄ ]⁺) and one other fragment ([MH-H₂O]⁺) were evident (FIGS. 1, 2 and 3). This actually allowed the presenceor absence of a tag to be determined by the observation of two signals,which removes any possible ambiguity when analyzing a more complexmixture. Approximately equal amounts of the three tags were then mixedand analyzed by PCIMS (FIG. 5). Again, the two signals corresponding toeach separate tag could easily be distinguished.

Tag 4 was now transformed into the diazoketone precursor 8 (Scheme 9),then attached to Tentagel resin as 9 (Scheme 12). One bead of the 9 wassubsequently removed and 4 oxidatively released using ceric ammoniumnitrate. PCIMS analysis again clearly showed the presence of tag 4.

In summary, the set of tags 4, 11 and 13 of the same structure, butdifferent molecular weights were synthesized. All were easily detectedsimultaneously in a mixture by PCIMS. The small amount of 4 releasedfrom a single bead of Tentagel resin used in combinatorial librarysynthesis is detectable by PCIMS. MS is a viable and sensitive detectionmethod for tags, and can be used as the basis for an encoding scheme ofa combinatorial library.

Analysis of 4, 11 and 13 by PCIMS was obtained using a reagent gasmixture of 1% NH₃ in CH₄.

(2). To a solution of 11.1 mL (125 mmole, 5.00 eq.) of 1,4-butanediol(1), 6.97 mL (50.0 mmole, 2.00 eq.) of Et₃ N and 0.153 g (1.25 mmole,0.05 eq.) of 4-dimethylaminopyridine in dry CH₂ Cl₂ (100 mL) at 0° C.under Ar, was added 3.88 g (25.0 mmole, 1.00 eq.) of 97%tert-butyldimethylsilyl chloride. The resulting solution was stirred at0° C. for 15 min, then 25° C. for 16 hours. The reaction was thendiluted with CH₂ Cl₂ (250 mL) and washed with 1 M HCl (100 mL),saturated aq. NaHCO₃ (100 mL) and H₂ O (100 mL), then dried (MgSO₄).Removal of the volatiles in vacuo gave the crude product 2 as an oil.

(3). To a solution of ˜10.0 mmole of crude alcohol 2, 1.93 g (10.5mmole, 1.05 eq.) of pentafluorophenol and 2.89 g (11.0 mmole, 1.10 eq.)of triphenylphosphine in dry CH₂ Cl₂ (40 mL) at 0° C. under Ar, wasadded 1.73 mL (11.0 mmole, 1.10 eq.) of diethyl azodicarboxylate. Theresulting orange solution was stirred at 0° C. for 5 min, then 25° C.for 15 hours. The reaction was then diluted with CH₂ Cl₂ (250 mL) andwashed with saturated aq. Na₂ CO₃ (100 mL), saturated aq. NH₄ Cl (100mL) and H₂ O (100 mL), then dried (MgSO₄). Removal of the volatiles invacuo and purification by flash chromatography (0-20% EtOAc in hexanes)gave the product 3 as an oil.

(4). To a solution of 1.85 g (5.00 mmole, 1.00 eq.) of silyl-protectedalcohol 3 in THF (20 mL) at 25° C., was added 10.0 ML (10.0 mmole, 2.00eq.) of a 1.0 M solution of tetrabutylammonium fluoride in THF. Theresulting orange solution was stirred at 25° C. for 4 hours. Removal ofthe volatiles in vacuo and purification by flash chromatography (20-40%EtOAc in hexanes) gave 1.10 g (86%) of the product 4 as an oil.

(5). To a solution of 0.800 g (3.125 mmole, 1.00 eq.) of alcohol 4,0.569 g (3.125 mmole, 1.00 eq.) of methyl vanillate and 0.984 g (3.75mmole, 1.20 eq.) of triphenylphosphine in dry CH₂ Cl₂ (20 mL) at 0° C.under Ar, was added 0.591 mL (3.75 mmole, 1.20 eq.) of diethylazodicarboxylate. The resulting pale yellow solution was stirred at 0°C. for 5 min, then 25° C. for 19 hours. The reaction was then dilutedwith CH₂ Cl₂ (100 mL) and washed with 1 M NaOH (50 mL), saturated aq.NH₄ Cl (50 mL) and H₂ O (50 mL), then dried (MgSO₄). Removal of thevolatiles in vacuo and purification by flash chromatography (20% EtOAcin hexanes) gave the product 5 as an oil.

(6). To a solution of 3.125 mmole of ester 5 in THF (12 mL) was added1.31 g (31.3 mmole, 10.0 eq.) of lithium hydroxide monohydrate. MeOH (24mL) was added to the resulting suspension to form a solution, which wasstirred at 25° C. for 1 hours, then refluxed for 1 day. Volatiles wereremoved in vacuo, and 1 M HCl then added until solution was ˜pH 1. Thewhite precipitate of product which formed was collected and dried togive 0.968 g (76%--2 steps) of 6.

(7). To 0.968 g (2.38 mmole, 1.00 eq.) of carboxylic acid 6 under Ar,was added 2.43 mL of thionyl chloride. The resulting suspension wasrefluxed for 1.5 hours, after which time a yellow solution had formed.Volatiles were removed in vacuo, and the resulting residue azeotropedthree times with toluene to give the product 7 as colorless crystals.

(8). To a solution of 2.38 mmole of acid chloride 7 in 1:1 THF:MeCN (20mL) at 0° C. under Ar, was added 1.16 mL (8.33 mmole, 3.50 eq.) of Et₃ Nfollowed by 3.57 mL (7.14 mmole, 3.00 eq.) of a 2.0 M solution of(trimethylsilyl)diazomethane in hexanes. The resulting yellow solutionwas stirred at 0° C. for 1 h, then 25° C. for 1 day. The reaction wasdiluted with EtOAc (150 mL) and washed with saturated aq. NaHCO3 (2×75mL) and saturated aq. NaCl (2×75 mL), then dried (MgSO₄). Removal of thevolatiles in vacuo gave the crude product 8 as an oil.

(11). Using commercially available 1,4-butanediol-2,2,3,3-d₄ (10) inplace of 1, and an analogous procedure to that described for thetransformation 1 into 4, 11 was obtained in 41% yield over three steps.

Using commercially available 1,4-butanediol-2,2,3,3,4,4-d₈ (10) in placeof 1, and an analogous procedure to that described for thetransformation 1 into 4, 13 was obtained in 42% yield over three steps.

Tag 4 was introduced onto the solid support using 5 to 50% (w/w vs.resin) precursor diazoketone 8 to give 9 by essentially the sameprocedure given in Example 4; the Hetero Diels-Alder library. Tag 4 wasalso subsequently removed from 9 by essentially the same procedure asStep G in Example 4.

The diazoketones corresponding to tags 11 and 13 are used to introducethese tags onto the solid support, so that along with 8, they yieldmembers of a binary encoding set. ##STR51##

It is evident from the above description that the subject inventionprovides a versatile, simple method for identifying compounds, where theamount of compound present precludes any assurance of the ability toobtain an accurate determination of its reaction history. The methodallows for the production of extraordinarily large numbers of differentproducts, which can be used in various screening techniques to determinebiological or other activity of interest. The use of tags which arechemically inert under the process conditions allows for greatversatility in a variety of environments produced by the varioussynthetic techniques employed for producing the products of interest.The tags can be readily synthesized and permit accurate analysis, so asto accurately define the nature of the composition.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

What is claimed is:
 1. A method of recording a reaction history of asolid support which comprises:a. reacting the support with a firstreagent under a first reaction condition; b. attaching to the support atleast a first and a second identifier, each having a non-sequencabletag, which tags differ each from the other, the combination of whichtags records the first reagent or first reaction condition; andthereafter c. reacting the support with a second reagent under a secondreaction condition; and d. attaching to the support at least a third anda fourth identifier each having a non-sequencable tag, which tags differeach from the other and from the first and second tags of (b), thecombination of which tags records the second reagent or second reactioncondition,wherein a compound is synthesized by a process comprising step(a) and step (c).
 2. The method of claim 1, wherein each tag of thesecond plurality is attached to the solid support through other than atag of the first plurality.
 3. The method of claim 2, wherein each tagis attached through a detachable linker so that the tags of theidentifiers are detachable from the support and, when detached, areseparable from each other.
 4. The method of claim 3, wherein thedetachable linker comprises an orthomethoxyphenyl ether.
 5. The methodof claim 3, wherein each tag, after detachment, comprises not more than100 atoms, other than hydrogen atoms.
 6. The method of claim 3, whereineach tag, after detachment, comprises not more than 60 atoms, other thanhydrogen atoms.
 7. The method of claim 3, wherein each tag comprises ahaloalkyl or haloarylalky moiety.
 8. The method of claim 1, wherein thereagent of each stage of the reaction history is recorded by a codeconsisting of a tag attached to the support or a combination of tagsattached to the support.
 9. The method of claim 1, wherein each tag isattached through a detachable linker so that the tags of the identifiersare detachable from the support and, when detached, are separable fromeach other.
 10. The method of claim 9, wherein the detachable linkercomprises an orthomethoxyphenyl ether.
 11. The method of claim 9,wherein each tag, after detachment, comprises not more than 100 atoms,other than hydrogen atoms.
 12. The method of claim 9, wherein each tag,after detachment, comprises not more than 60 atoms, other than hydrogenatoms.
 13. The method of claim 9, wherein each tag comprises a haloalkylor haloarylalky moiety.
 14. A method of recording a reaction history ofa solid support, which comprises:a. forming a plurality of first groupsof solid supports, which groups together comprise at least 100individual solid supports; b. reacting each support of each group of thefirst plurality with a first reagent under a first reaction condition;c. attaching to each support of a group of the first plurality at leasta first and a second identifier each having a non-sequencable tag, whichtags differ each from the other, the combination of which tags recordsthe first reagent or first reaction condition of the group; andthereafter d. mixing the supports of the first groups, each group withthe others, and dividing the mixture into a plurality of second groups,which groups together comprise at least said 100 individual solidsupports; e. reacting each support of each group of the second pluralitywith a second reagent under a second reaction condition; and f.attaching to each support of a group of the second plurality at least athird and a fourth identifier, each having a non-sequencable tag, whichtags differ each from the other and from the first and the second tagsof (c), the combination of which tags records the second reagent orsecond reaction condition of the group,wherein a compound is synthesizedby a process comprising step (b) and step (e).
 15. The method of claim14, wherein each tag of the second plurality is attached to the solidsupport other than through a tag of the first plurality.
 16. The methodof claim 14, wherein the reagent of each stage of the reaction historyfor each support is recorded by a code consisting of a tag attached tothe support or a combination of tags attached to the support.
 17. Themethod of claim 14, wherein each tag is attached through a detachablelinker so that the tags of the identifiers are detachable from thesupport and, when detached, are separable from each other.
 18. Themethod of claim 17, wherein the detachable linker comprises anorthomethoxyphenyl ether.
 19. The method of claim 17, wherein each tag,after detachment, comprises not more than 100 atoms, other than hydrogenatoms.
 20. The method of claim 17, wherein each tag, after detachment,comprises not more than 60 atoms, other than hydrogen atoms.
 21. Themethod of claim 17, wherein each tag comprises a haloalkyl orhaloarylalkyl moiety.
 22. A method of recording a reaction history of asolid support which comprises:a. reacting the support with a firstreagent under a first reaction condition; b. attaching to the support afirst identifier having a non-sequencable tag, which tag, alone or incombination with one or more other tags, records the first reagent orfirst reaction condition; and thereafter c. reacting the support with asecond reagent under a second reaction condition; and d. attaching tothe support a second identifier having a non-sequencable tag, which tag,alone or in combination with one or more other tags, records the secondreagent or second reaction condition,wherein a compound is synthesizedby a process comprising step (a) and step (c), wherein the tag of thefirst identifier and the tag of the second identifier differ each fromthe other, and wherein each tag is attached to the solid support throughother than the compound via a detachable linker so that the tag of thefirst identifier and the tag of the second identifier are detachablefrom the support and, when detached, are separable from each other. 23.The method of claim 22, wherein the second tag is attached to the solidsupport through other than the first tag.
 24. The method of claim 22,wherein the reagent of each stage of the reaction history for eachsupport is recorded by a code consisting of a tag attached to thesupport or a combination of tags attached to the support.
 25. The methodof claim 22, wherein the detachable linker comprises anorthomethoxyphenyl ether.
 26. The method of claim 22, wherein each tag,after detachment, comprises not more than 100 atoms, other than hydrogenatoms.
 27. The method of claim 22, wherein each tag, after detachment,comprises not more than 60 atoms, other than hydrogen atoms.
 28. Themethod of claim 22, wherein each tag comprises a haloalkyl orhaloarylalkyl moiety.
 29. A method of recording a reaction history of asolid support which comprises:a) forming a plurality of first groups ofsolid supports, which groups comprise together at least 100 individualsolid supports; b) reacting each support of each group of the firstplurality with a first reagent under a first reaction condition, whereinthe reagent or the reaction condition for each of the first groupsdiffers from the reagent or reaction condition for all other firstgroups; c) attaching to each support of each group of the firstplurality an identifier having a non-sequencable tag, which tag alone orin combination with one or more other tags records the first reagent orfirst reaction condition; and thereafter d) mixing the supports of thefirst groups, each group with the others, and dividing the mixture intoa plurality of second groups, which groups together comprise at leastsaid 100 individual solid supports; e) reacting each support of eachgroup of the second plurality with a second reagent under a secondreaction condition, wherein the reagent or the reaction condition foreach of the second groups differs from the reagent or reaction conditionfor all other second groups; and f) attaching to each support of eachgroup of the second plurality a second identifier having anon-sequencable tag, which tag alone or in combination with one or moreother tags records the second reagent or second reactioncondition,wherein the tags of each group of the first plurality ofgroups and second plurality of groups differ from the tags of all othergroups, wherein each tag is attached through a detachable linker, sothat the tags of the first plurality and the tags of the secondplurality are detachable from the support and, when detached, areseparable from each other, and wherein a compound is synthesized by aprocess comprising step (b) and step (e).
 30. The method of claim 28,wherein each tag of the second group is attached to the solid supportthrough other than a tag of the first group.
 31. The method of claim 28,wherein the reagent of each stage in the reaction history of eachsupport is recorded by a code consisting of a tag attached to thesupport or a combination of tags attached to the support.
 32. The methodof claim 30, wherein the detachable linker comprises anorthomethoxyphenyl ether.
 33. The method of claim 30, wherein each tag,after detachment, comprises not more than 100 atoms, other than hydrogenatoms.
 34. The method of claim 30, wherein each tag, after detachment,comprises not more than 60 atoms, other than hydrogen atoms.
 35. Themethod of claim 30, wherein each tag comprises a haloalkyl orhaloarylalkyl moiety.